260/280: assess the purity of DNA and RNA
~1.8 is generally accepted as “pure” for DNA 260/230 Ratios: measure of nucleic acid purity...often higher than the respective 260/280 values.
~2.0-2.2. PfDXR extinction coefficient: ~39162.5 M-1 cm-1 PfDXR molecular weight: 47206.6 Da
Useful Libraries (Docking programs indicate compounds may be of use in these libraries)
cb_306_3D.sdf
ChemBridge-3D
MayBridge50k_3D
MW-set_3D
HF9_Plates_1um3D_catnum.sdf
Screened Libraries (TO DO)
chembIntd (Skip - paper indicates compounds do not target DXR)
MAR3D7 (Skip - paper indicates compounds do not target DXR)
Microsets
January 21, 2013
PreFPLC PfDXR concentrated to 0.5 ml - takes about 50 min
yield: 3.39 mg
Used Umeda Buffers (all include 2 mM DTT - reducing agent) for protein purification
Also learned what happens when the pH drops below 7 in protein solutions and when you fail to remove the sticker from the bottom the SDS Page Gel. Week 13 Freebie from Journal Club Team B victory
Week 12 November 30, 2012 Enzymatic Assay not promising
Several enzyme assays (varying in room temp. vs 37 degrees Celsius and varying in duration from 5-15 min) were performed. Both snap frozen and -20 stocks of PfDXR were used. Absorbance of NADPH was measured at 340 nm. All trials revealed a constant gradient for NAPDH absorbance, indicating that NADPH was not being oxidized.
The obvious conclusion is that the PfDXR enzyme we synthesized is not functional. However, purification, characterization and concentration were all completed within a span of 12 hours, so the probability the enzyme denatured is low.
An alternative is to modify the assay parameters. Based on literature searches, the optimum temp. for PfDXR activity is 37 degrees Celsius. Most of the trials were done at room temp., which may explain why the absorbance was flat lined. For the two assays done at 37 degrees Celsius, one was done for 5 min. only while the other was "oversaturated" with enzyme - therefore, changing the optimal conc. of buffer and reagents. (NEED to verify the last sentence with data on computer in lab).
The remaining option is to redo the assay at 37 degrees Celsius with the following changes.
1. 5 min incubation with reagents and buffer at 37 degrees Celsius in PCR thermocycler.
2. Add PfDXR enzyme and obtain NADPH absorbance at 340 nm.
3. Incubate at 37 degrees Celsius for 10-20 min.
4. Read the absorbance value at 340 nm again.
If the above steps do not reveal a depletion of NADPH (i.e. 0 absorbance at 340 nm after 10-20 min), then all hope is lost - the enzyme is inactive. November 28, 2012 Prepared enzyme assay reagents
NADPH (cofactor) and DXP (substrate) were solubilized in 0.01 M NaOH and pure water, respectively to obtain 192 5 mM NADPH aliquots (5 enzyme assays each) and 175 12 mM DXP aliquots (1 enzyme assay each). Tris-HCl was diluted to 125 mM.
November 27, 2012 Protein sonication (lysing), purification (extraction), characterization (verification), and concentration
PfDXR homodimer is present in fraction samples 25 through 29. Presence can be verified by molecular standards (77 kDa is the small bump near fraction samples 29 through 31). PfDXR monomer is approx. 47 kDa (dimer will be approx. 94 kDa).
Rescaled FPLC graph (same as above). Small bump at fractions 31 through 34 may indicate monomer - standard at fractions corresponding to 33 through 36 is approx. 33 kDa.
November 26, 2012 IPTG induction, 20 hour overnight expression
112612 - ??. Dr B Week 11 November 21, 2012
Ranking of Libraries by top scoring ligands (11/21/12)
1. ChemBridge-3D
2. HF9_180_Plates_1um3D_catnum
3. MayBridge50k_3D
4. MW-set_3D
5. cb_306_3d
6. NIH_ClinCol3Ded
7. Fragment-set_3D
8. HF9PlatesPlates5_9
Top Ten Ligand List did not change from Week 8 results. However, MayBridge50k_3D entered the top 20 list and MW-set_3D entered the top 30 list.
Top Compound No. 1 7576671 docked in active site of PfDXR
7576671 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
Top Compound No. 3 7969923 docked in active site of PfDXR
7969923 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
Top Compound No. 4 5583722 docked in active site of PfDXR
5583722 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
FOM docked in active site of PfDXR
FOM shown as sticks (Carbon purple).
MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
November 20, 2012
We went ahead and transformed colonies 5 and 15 into BL21 (DE3) competent cells. Also, we started a 320mL culture of colony 5 for Midiprep. Will spin down and Midiprep tomorrow.
November 19, 2012 DNA sequencing shows positive clones for submitted samples
Miniprepped plasmid samples from colonies 5,6,15,16, 21 & 22. A BLAST comparison indicated positive clones for every colony without mutations.
Week 10 - good - Dr. B 11/19/12 November 18, 2012 RE Digest with HincII enzyme to check for positive clones
Virtual Gel of PfDXRL75 - positive clones on gel image should have bands at sizes indicated above.
Gel Image of RE Digest of PfDXR in pNIC-Bsa4 with HincII for Colonies 1-8
KRAB_PfDXR_REDigest_1t8_111812.png
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: Colony 1 - not likely to be full insert
Lane 4: Colony 2 - not likely to be full insert
Lane 5: Colony 3 - not likely to be full insert
Lane 6: Colony 4 - not likely to be full insert
Lane 7: Colony 5 - positive clone (sequenced)
Lane 8: Colony 6 - positive clone (sequenced)
Lane 9: Colony 7 - likely a positive clone (did not sequence)
Lane 10: Colony 8 - likely a positive clone (did not sequence)
Gel Image of RE Digest of PfDXR in pNIC-Bsa4 with HincII for Colonies 9-16
KRAB_PfDXR_REDigest_9t16_111812.png
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: Colony 9 - likely a positive clone (did not sequence)
Lane 4: Colony 10 - likely a positive clone (did not sequence)
Lane 5: Colony 11 - likely a positive clone (did not sequence)
Lane 6: Colony 12 - likely a positive clone (did not sequence)
Lane 7: Colony 13 - not likely to be full insert
Lane 8: Colony 14 - not likely to be full insert
Lane 9: Colony 15 - positive clone (sequenced)
Lane 10: Colony 16 - positive clone (sequenced)
Gel Image of RE Digest of PfDXR in pNIC-Bsa4 with HincII for Colonies 17-24
KRAB_PfDXR_REDigest_17t24_111812.png
Lane 1: Skip
Lane 2: 1 kb Ladder
Lane 3: Colony 17 - likely a positive clone (did not sequence)
Lane 4: Colony 18 - likely a positive clone (did not sequence)
Lane 5: Colony 19 - likely a positive clone (did not sequence)
Lane 6: Colony 20 - not likely to be a positive clone
Lane 7: Colony 21 - positive clone (sequenced)
Lane 8: Colony 22 - positive clone (sequenced)
Lane 9: Colony 23 - not likely to be a positive clone
Lane 10: Colony 24 - not likely to be a positive clone
November 17, 2012 Master Plate Protocol completed on 24 colonies
November 16, 2012 Cut pNIC and Cloning and Transformation
cut pNIC-Bsa4 at BsaI sites - combined two samples (starting conc. 82.4 ng/ul)
November 15, 2012 More PCR from plasmid combined 5 samples (20 cycles each)
November 14, 2012 Back to cloning part 2
Transformation was not successful November 13, 2012 Back to cloning
We used the new L75 forward primer to do PCR.
Lane 1 & 5-10: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: 10 ul PfDXRL75 (~1300 bp) - used 1 ng in 50 ul reaction volume, 20 cycles
Lane 4: 10 ul PfDXRL75 (~1300 bp) - used 5 ng in 50 ul reaction volume, 20 cycles
PCR Clean Up was not as fruitful (21.9 ng/ul), but satisfactory
Week 9 November 08, 2012 PCR from primary mix- used wrong forward primer
supersec: 20 sec annealing
kr2: 1 min annealing
Lane 3: 1A primary smear -primary mix made in June kr2 protocol
Lane 4: 1B primary smear -primary mix made in July kr2 protocol
Lane 3: 1A primary smear - primary mix made in June supersec protocol
Lane 4: 1B primary smear - primary mix made in July supersec protocol
Lane 5: 2A secondary - kr2 protocol
Lane 6: 2B secondary - kr2 protocol
secondary from 1B
Lane 1&6: 100 bp Ladder
Lane 2: 2B secondary - kr2 protocol
Lane 3: 2B secondary - kr2 protocol
Lane 4: 2B secondary - supersec protocol
Lane 5: 2B secondary - supersec protocol
PCR^2 from 2B secondary -kr2 protocol
November 07, 2012 Combined efforts no go - used wrong forward primer
Andrew, Alex and I gave it another attempt at PCR from the plasmid DNA.
Lane 1: 1kB DNA Ladder
Lane 2-5: Alex's vain attempt using modified Aptamer PCR protocol on plasmid DNA (15 cycles)
Lane 6: Kaarthik's vain attempt using custom protocol on plasmid DNA (30 cycles)
Lane 7: Kaarthik's vain attempt using primary mix DNA with custom protocol. (30 cycles)
Lane 8: Andrew's vain attempt using Aptamer protocol (no modifications) on plasmid DNA (15 cycles)
Lane 9-12: Recheck of secondary PCR with new primers (20 cycles only).
Looks like we made a new DNA ladder!
We will remake the primary DNA and use the new forward to make secondary PCR DNA. November 04 and 05 and 06, 2012 PCR Party no go - used wrong forward primer
PCR on the plasmid DNA was attempted several times to no avail. I went back to the primary 1A mix (first mix made in the June 2012) and used it as template with the new forward primer. The secondary DNA output appeared to be the correct size in bp, but PCR^2 from this secondary resulted in smaller amplicon length.
After these successive failures, I will do a dpnI digest of the 17 ng/ul DNA and anneal/transform. The only downside is that the gene appears to be near 1500 bp (suggesting I used the wrong forward primer) but the gel can be deceiving.
Lane 1: 1kB DNA Ladder
Lane 2-5: Secondary PCR^2 - wrong gene size
Lane 6-12: Secondary PCR^2 - no DNA product
Lane 1: 1kb DNA Ladder
Lane 2: 1A Primer smear
Lane 3-6: Secondary PCR from 1A primary
Lane 7-10: Another attempt at DNA amplification from plasmid DNA
November 02, 2012 PCR from plasmid DNA - used wrong forward primer
No PCR product - will probably need to adjust timing of denaturation and annealing steps
-maybe try diluting stock plasmid directly (add 500 ul Tris HCl to original amount).
-or do three fold dilution from 90 ng/ul to 45 ng/ul to 22.5 ng/ul to 11.25 ng/ul
November 01, 2012 PCR from plasmid DNA. Incorrectly followed right protocol - used wrong forward primer
Lane 1 & 8-10: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: PCR using overlap extension conditions - 84 ng of plasmid DNA (10 ul added to gel)
Lane 4-7: PCR using plasmid DNA conditions
There was no product in lanes 4-7.
Mistakes made:
1. Used MgSO4 instead of MgCl2
2. Used PCR-Overlap thermocycling conditions instead of recommended protocol.
October 31, 2012 PCR^2 from plasmid DNA. Followed wrong protocol - used wrong forward primer
DNA replication results were not promising. We used nearly 84 ng (the KOD info sheet recommends 1 ng only of plasmid DNA) so we need to modify the cycling conditions.
Week 8 Official No Wet Lab Week
No significant work done in lab.
All of the jobs submitted to Maestro for 2D to 3D conversion of the chembIntd and MAR3D7 libraries failed to complete. One job succesfully ran but prematurely exited with a fatal message (FATAL m2io_get_number_blocks(): error getting number of blocks in file) while the other 4 did not begin.
Virtual screening was completed on all of the proposed libraries. Some results follow.
Top Ten Ligands after 2 Runs of Screened Libraries 10/28/12
Ranking of Libraries by top scoring ligands
1. ChemBridge-3D
2. HF9_180_Plates_1um3D_catnum
3. cb_306_3d
4. NIH_ClinCol3Ded
5. Fragment-set_3D
6. HF9PlatesPlates5_9
Top Compound No. 2 RJC00044SC docked in active site of PfDXR
Validation docking was also completed (Autoscale = 2; comparable to above top ten) on known inhibitor Fosmidomycin (FOM)
While FOM has a smaller Score than the top ten compounds, it has the second best S(hbond) and the best S(metal) score.
Original FOM docked in the active site of PfDXR
Original FOM aligned with Validation Docking Pose No. 4 FOM
The Validation indicates that Virtual Screening may provide reliable results for identifying inhibitors.
Week 7
102112- Yeah - keep up the good work. Dr. B October 19, 2012 Midiprepped PfDXR
Andrew spun down a culture of E. Coli with the recombinant DNA and the DNA was extracted with the Qiagen set.
October 17, 2012 DNA Sequencing Results
Sample 2A5 has the full coding sequence in the pNIC-Bsa4 vector.
The Gaps included 2 deletions approximately 100 bp before the start codon which are likely false positives and 2 deletions around the 1050-1060 bp no. range and are false positives since these mutations were not present in the reverse sequence.
There were no gaps in this read of the target coding sequence. There were several "N" - the actual bp at these locations was verified by reading the peaks displayed in the chromatogram and comparing base location to the forward read.
October 16, 2012 DNA Sequencing Results
The predictions about which samples had the insert were wrong. Neglecting that, there were two positive clones in the sequencing results: 1A2 and 2A3. 1A3 had about 4 deletions and 2A3 had 1 deletion only. There may be more deletions (or other mutations) since we only sent the forward pLIC primer with the samples. Since we know the 2A samples are reliable, we will submit the following samples to sequencing:
2A3 with pLIC-rev, oligoprimer no. 1, oligoprimer no. 38
2A1 with pLIC-for
2A4 with pLIC-for
2A5 with pLIC-for and pLIC-rev
October 15, 2012 DNA Sequencing Submission
The following samples were sent to sequencing:
1A3 1A2 1A5 1A6 1B2 2A3 2A5 2B6
All of them were submitted with the pLIC-for primer.
101612 - Kaarthik - ok good deal. Hopefully the DNA sequencing works. -- Dr. B
Week 6 October 13, 2012 Restriction Enzyme Digest of miniprep samples with HincII
The following restriction enzyme digest indicated the successful transformation of E. Coli DH5a with recombinant DNA pNIC-Bsa4 PfDXR construct, albeit only in a few cases. No DNA ladder was added to the agarose gel because 12 samples were added to each agarose gel (with 12 lanes).
HincII RE Digest of Samples from A & B plates containing 249.2 ng/ul insert.
Lane 1. 1A1 - likely insert
Lane 2. 1A2 - unknown 10/16/12 INSERT: several deletions with pLIC-for
Lane 3. 1A3 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 4. 1A4 - no digest by HincII
Lane 5. 1A5 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 6. 1A6 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 7. 1B1 - likely insert
Lane 8. 1B2 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 9. 1B3 - no digest by HincII
Lane 10. 1B4 - likely insert
Lane 11. 1B5 - unknown
Lane 12. 1B6 - no digest by HincII
The digest was repeated for the A & B plates containing the 339.1 ng/ul insert
HincII RE Digest of Samples from A & B plates containing 339.2 ng/ul insert
Lane 1. 2A1 - more than likely insert 10/17/12: INSERT: Several deletions in Insert
Lane 2. 2A2 - no digest by HincII
Lane 3. 2A3 - more than likely insert 10/16/12: INSERT: only one deletion present with pLIC-for 10/17/12: Several deletions in Insert
Lane 4. 2A4 - more than likely insert 10/17/12: INSERT: Several deletions in Insert
Lane 5. 2A5 - confirmed insert 10/16/12: INSERT: MAGIC
Lane 6. 2A6 - no digest by HincII
Lane 7. 2B1 - no DNA
Lane 8. 2B2 - no digest by HincII
Lane 9. 2B3 - no digest by HincII
Lane 10. 2B4 - no DNA
Lane 11. 2B5 - no digest by HincII
Lane 12. 2B6 - more than likely insert 10/16/12: NOT AN INSERT: tail end
For the above gel, we cannot conclude if the PfDXR gene is present because a successful cut of the recombinant DNA should produce 5 cuts, with 2 cuts visible and 3 cuts faded - see Sept. 14, 2012 or Sept. 24, 2012).
We made two mistakes in this trial. One, we should have added the DNA Ladder to make comparisons reliable and conclusions easier to validate. Second, we should have not run the gel for 45 minutes - 35 minutes at 110 V may be ideal for a RE Digest, especially since a digest with HincII will produce several DNA strands below 500 bp.
Another conclusion is to decrease the digest time to 1 hour since star activity is visible in both gels above. We will submit the following samples with the pNIC-for primer to DNA Sequencing:
1A3 1A2 1A5 1A6
1B2
2A3 2A5
2B6
October 12, 2012 Miniprep RE Digest
Since there were 24 samples, only measurement (1A1) was uploaded.
The 2B samples may be of low purity because we left ethanol solution in the sample tubes before proceeding to elution the next morning.
Colonies grew overnight on the four plates. We chose 24 colonies (6 from each plate) to continue for miniprep.
October 10, 2012 Cohesive End Generation, Annealing and Transformation
We ran a gel with 1 ul of the 340 ng/ul PfDXR cleaned up and 1 ul of the accepting vector cut with BsaI.
Gel indicating presence of 1.5kB PfDXR gene and cut pNIC-Bsa4
Lane 1: Skip
Lane 2: 1 kB DNA Ladder
Lane 3: PfDXR cleaned up (339.1 ng/ul)
Lane 4: cut pNIC-Bsa4
Lane 5-10: Skip
We also did cohesive end generation with the new T4 polymerase, dGTP and dCTP (purchased in early October). We did the annealing and transformation step verbatim. Four plates were made (No. 1 contained the insert that originally came from the 249.2 concentrated stock and No. 2 contained the insert from the 339.1 concentrated stock) and one set of plates contained 2 ul of accepting vector + 4 ul of insert and the other set of plates contained 48 ul of accepting vector and 16 ul of insert.
October 09, 2012 pNIC-Bsa4 Midiprep, pNIC-Bsa4 vector preparation
We midiprepped pNIC-Bsa4 according to the Qiagen protocol. We doubled the buffer volume (i.e. 6 ul to 12 ul) as was suggested in the booklet to increase DNA yield, but the result of this is uncertain because we ended with a low concentration of the vector - 51.5 ng/ul. The yield may also be low because we did not adhere strictly to time constraints (i.e. incubation times were longer).
Nanodrop of pNIC-Bsa4 eluted in 5 mM Tris HCl. Concentration was 51.5 ng/ul, 260/280 at 1.91, 260/230 at 2.29.
We also cut a 82 ng/ul pNIC-Bsa4 vector with BsaI. The results are promising. In the past, we let the RE Digest go for 2.5 hours, but we increased the time to 3 hours in this instance.
Nanodrop of pNIC-Bsa4 cut with BsaI eluted in 10 mM Tris HCl. Concentration was 71.7 ng/ul, 260/280 at 1.93, 260/230 at 2.30
October 08, 2012 Secondary PCR, PCR^2
We successfully made high concentrations of the PfDXR encoding gene from PCR. We deviated from standard protocol in that we decreased the PCR^2 thermocycling to 25 cycles in the hopes of minimizing mutations.
Verification of Secondary PCR
Lane 1: Skip
Lane 2: 1kB DNA Ladder (3 ul)
Lane 3: Secondary PCR from primary mix C prepared in the beginning of July (5 ul)
Lane 4-10: Skip
We used the KOD polymerase from 09/12/12 - we made sure to immediately transfer the final 50 ul sample to the thermocycler once the KOD was added. This was likely the critical step that we had failed to observe in last week's PCR attempts.
We then repeated the thermocycling with the secondary PCR template from above but did 25 cycles only.
Verification of PCR^2
Lane 1: Skip
Lane 2: 1kB Ladder
Lane 3: Secondary PCR^2 from primary C
Lane 4: Secondary PCR^2 from primary C
Lane 5: Secondary PCR^2 from primary C
Lane 6: Secondary PCR^2 from primary C
Lane 7-10: Skip
Since we hurried, the gel image is poor - we halted 20 minutes prematurely. Fortunately, the 1.5 kB band is visible in Lanes 3,4 and 5. The unevenness of the brightness in these lanes may be attributed to improper mixing of reagents prior to adding KOD polymerase to the master mix.
We then cleaned up the sample and eluted in 10 mM Tris-HCL.
Nanodrop measurement of 2 ul of PfDXR eluted in 50 ul of 10 mM Tris HCl. Concentration at 249.2 ng/ul, 260/280 at 1.89 and 260/230 at 2.42.
The absorbance values are reasonable and the concentration broke a personal record set on July 09, 2012.
We checked the clean up below.
Verification of PfDXR Gene after clean up
Lane 1: Skip
Lane 2: 1kB DNA Ladder
Lane 3: 2 ul of 249.2 ng/ul PfDXR cleaned up
Lane 4-10: Skip
No tail end primers are visible - indicating minimal contamination.
We repeated the thermocycling to produce more PCR^2 to ensure we have enough for cloning. While the absorbance values are in range and the concentration is high, we will need to gel check the sample later to verify the presence of the PfDXR gene.
Nanodrop measurement of 2 ul of PfDXR (Sample No. 2) eluted in 50 ul 10 mM Tris HCL. Concentration at 339.1 ng/ul. 260/280 at 1.85, 260/230 at 2.27.
We will continue with cloning this week.
Week 5 100912 - look great. Keep on truckin' - DR. B
October 05, 2012 Verification of plates, PCR, Gel Check
The VDS Staff plates made in September were okay. At least 50 colonies grew on the plate containing SCP1 in pET. On our transformation attempt, the cohesive end generation may have failed - explaining the lack of colonies on the plates.
We also tried secondary PCR twice - once with the Q5 polymerase and the other with Phusion. Both attempts failed; it is possible that the PCR samples were left on ice too long after the polymerase was added.
Gel Check to verify the presence of DNA in samples
We added 5 ul of sample to each lane
Lane 1: Skip
Lane 2: 1kB DNA Ladder
Lane 3: 1A - Primary prepared in Summer
Lane 4: 1B - Primary prepared in Summer
Lane 5: 1C - Primary prepared in Summer
Lane 6: alpha - Primary prepared in Fall (09/06/12)
Lane 7: gamma - Primary prepared in Fall (09/06/12)
Lane 8: Secondary prepared in Summer
Lane 9: gamma - Secondary prepared in Fall (from the Lane 7 sample)
Next week we will continue with making secondary PCR samples and attempt another transformation.
October 04, 2012 Unsuccessful transformation of pNIC-Bsa4:PfalDXR in DH5-alpha.
No colonies grew on the four LB+Kan+5% Sucrose plates. Other colleagues did not have success on the same batch of plates so we transformed another kanamyacin resistant vector and YopH to determine the efficacy of the plates.
We also prepared another 20 plates with LB+Kan.+5% Sucrose.
October 02, 2012 Annealing and Transformation Take 5, More PCR
The PCR Insert from October 01, 2012 was annealed with the cut pNIC-Bsa4, below. The RE Digest was done over 2.5 hours.
Nanodrop measure of pNIC-Bsa4 digested with BsaI. Concentration 39.1 ng/ul, 260/280 at 2.01 and 260/230 at 2.16.
We also completed several PCR trials. The four PCR^2 samples were done with new dilutions of the forward and reverse primers.
Agarose Gel containing cut pNIC-Bsa4, secondary PCR samples and PCR^2 samples.
Lane 1: 1 kB DNA Ladder
Lane 2: pNIC-Bsa4 Cut after PCR Clean Up
Lane 3: Secondary gamma - original mix from primary 09/04/12 (3 ul) 20 cycles
Lane 4: Secondary alpha - original mix from primary 09/04/12 (3 ul) 20 cycles
Lane 5: Secondary gamma - original mix from primary 09/06/12 (3 ul) 25 cycles
Lane 6: Secondary alpha - original mix from primary 09/06/12 (3 ul) 25 cycles
Lane 7: Skip
Lane 8: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 9: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 10: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 11: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 12 : Skip
We then cleaned up the PCR^2 samples from above. Unfortunately, the concentrations were too low for use and the samples were discarded. We performed two trials and deviated from the default protocol. Instead of combining all four PCR samples into one tube, we combined 2 samples into one tube and continued the clean up from there. As a result, only 100 ul of DNA sample was cleaned up in each trial.
We did another PCR starting from the secondary mix. 4 copies were made, but all were done with 21 cycles only to minimize the possibility of mutations. Decreasing the cycles may have some tradeoffs, including decreased final product yield and increased contamination (more oligoprimers).
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Skip
Lane 4: PCR^2 Sample 1 (3 ul)
Lane 5: Skip
Lane 6: PCR^2 Sample 2 (3 ul)
Lane 7: Skip
Lane 8: PCR^2 Sample 3 (3 ul)
Lane 9: Skip
Lane 10: PCR^2 Sample 4 (3 ul)
Lane 11: Skip
Lane 12: Skip
The difference in band intensity arises from unequal distribution of the master mix to the 4 aliquots. The gel also indicates the presence of a lot of smaller primers (tail end primers) in the PCR samples. This is likely a result of less cycling.
Interestingly, the concentration is comparatively high. The 260/230 absorbance ratio is less then preferred at 1.54
We ran 3 ul of the PCR cleanup Sample on a 1% agarose gel. Surprisingly, there does not seem to be a lot of contamination even though the nanodrop result indicated otherwise.
Lane 1: Skip
Lane 2: 1 kB DNA Ladder
Lane 3-9: Skip
Lane 10: Pfal DXR Clean up (3 ul)
1000112 - Kaarthik - any updates? Dr. B Week 4
September 28, 2012 DNA Sequencing Analysis for alpha 2, alpha 4, and alpha 12
From 16 colonies, 15 were successfully miniprepped. In the end, 3 colonies had the full coding DNA sequence, albeit with mutations. Most were deletions and are summarized below. The mutations were confirmed with DNA sequencing with five primers, pLIC-for, pLIC-rev, oligonucleotide 1, oligonucleotide 9, and oligonucleotide 38.
alpha 12, at least 7 deletions at bp 423, 424, 524, 1371, 1372, 1374, 1376
alpha 2, at least 7 deletions at bp 423, 424, 524, 1341, 1372, 1374, 1376
alpha 4, at least 2 deletions confirmed at bp 404, 566, one point mutation at bp 898 G replaced for A, another 3 potential mutations at 406, 407, 408 (2 sequencing results indicated deletions at these sites, while one did not).
Since the mutations occur in multiple regions, it is improbable we can do site-directed mutagenesis on these samples. We will continue from secondary PCR but decrease cycling to 20 cycles. This may decrease the number of copies with mutations.
September 24, 2012 Restriction enzyme Digest for 8 more colonies from alpha plate
We ran another digest on the 8 samples from Sept. 21, 2012. There was one positive clone, alpha-12, which was submitted for DNA Sequencing.
Lane 1: Skip
Lane 2-8: alpha n (where n begins at 5 and ends at 11)
Lane 9: alpha 12 pNIC-Bsa4 with Insert cut with HincII
Week 3
September 21 ,2012 - Kaarthik, looks good. Hopefully most of those indels won't turn out to be bad or else we can remove them with site-directed mutagenesis. -- Dr. B Blast Sequencing Results with custom oligo primers and growth of more colonies from the alpha plate.
The results from sequencing were not positive. A2-for corroborated deletions at 578, 579 and the insertion at 679 while A3-rev corroborated deletions at 578, 579, 679, 1525, 1526, 1528, and 1530. Both A2-for and A2-rev revealed no deletion at 750, however. NOTE: BP deletion sites are wrong. See Sept. 28 analysis for correct data. -- Sept. 28.
The results from the A4 set were more promising but both A4-for and A4-rev indicated deletions at 558, and 720. At 560,561 and 562 there were no deletions and 372 was not sequenced by either DNA sample.
However, in anticipation of unpromising Sequencing results, another 8 colonies were selected from the alpha plate to grow overnight on 9/20. The nanodrop concentrations after miniprep are shown below.
The concentrations are much lower than shown in previous trials with other colonies. The yield may have decreased since the colonies grew over one night, whereas the colonies from Sept. 14 grew over two nights. Another plausible reason is that the centrifuge time was decreased to 5 min (instead of 8 min in the September 14 trials). The next step is to pray that these samples have the complete coding DNA sequence of PfDXR and have a 100% identity match. We will do another restriction enzyme test with HincII before sending (any) positive clones to DNA Sequencing.
September 19, 2012 BLAST Sequencing Results
A2 pLIC-for 98% Max Ident.
A2 pLIC-rev 99% Max Ident.
A4 pLIC-for 98% Max Ident.
A4 pLIC-rev 99% Max Ident.
PfDXR Begins at base pair 155, ends at base pair 1621.
Insertions/Deletions:
A2-for, reliable from 134 to 982: 578 (deletion-C) 579 (deletion-G) 679 (insertion-C). Insertion at 679 not seen in A2-rev.
A2-rev, reliable from 741 to 1673: 750 (deletion-T) 1525 (deletion-C), 1526 (deletion-G), 1528 (deletion-G), 1530 (deletion-A). Deletion at 750 not seen in A2-for.
A4-for, reliable from 78 to 982: 372 (insertion C) 558 (deletion-T) 560 (deletion-C) 561 (deletion-A) 562 (deletion-C) 720 (deletion-C). Deletion at 720 IS seen in A4-rev.
A4-rev, reliable from 743 to 1683: No mutations.
Will send another sample to DNA Sequencing
A2-for: primer no. 9
A2-rev: primer no. 38
A4-for: primer no. 9
A4-rev: primer no. 38
Kaarthik - nice. - Dr. B 091812
September 17, 2012 DNA Sequencing
Submitted 7 samples to the ICMB Core DSF (500 ng DNA each).
Week 2
September 14 , 2012 Gel Analysis of Restriction Enzyme Digest (HincII) on pNIC-Bsa4 with Insert including controls
Lane 1: 1 kB DNA Ladder (5 ul)
Lane 2: Control: pNIC-Bsa4 Uncut
Lane 3: Control: pNIC-Bsa4 Cut with HincII
Lane 4: alpha-1 pNIC-Bsa4 with Insert Cut with HincII
Lane 5: alpha-2 pNIC-Bsa4 with Insert Cut with HincII
Lane 6: alpha-3 pNIC-Bsa4 with Insert Cut with HincII
Lane 7: alpha-4 pNIC-Bsa4 with Insert Cut with HincII
Lane 8: beta-2 pNIC-Bsa4 with Insert Cut with HincII
Lane 9: beta-3 pNIC-Bsa4 with Insert Cut with HincII
Lane 10: tau-1 pNIC-Bsa4 with Insert Cut with HincII
The Restriction Enzyme Digest positively confirms that the full Insert has been annealed to the accepting vector. Unfortunately, the gel was not run long enough (20 minutes, 130 V), resulting in a lackluster image. Fortunately, the digests on the Control (Lane 3) and the samples are distinguishable. There should be 3 cuts above 500 bp in the Control vector (Lane 3) according to the virtual digest available from NEBcutter. The top most band on Lane 3 may be the result of DNA sample from Lane 2 or DNA that was not cut. Meanwhile, it is also theorized that the pNIC-Bsa4 with insert will have 5 cuts, source: virtual digest from NEBcutter. What was expected correlates to the actual result, especially in alpha-2 and alpha-4 (Lane 4 and Lane 6, respectively). Overall, difference in band sizes indicates a successful transformation of pNIC-Bsa4. The next step is to verify the quality of the DNA amplification via DNA Sequencing.
pNIC-Bsa4 No Insert Virtual Digest (NEBcutter 2)
pNIC-Bsa4 DOXP Virtual Digest (NEBcutter 2)
Nanodrop of Miniprepped pNIC-Bsa4 Insert
Originally, we grew the E. Coli in 8 separate transformation tubes (alpha-1, alpha-2, alpha-3, alpha-4, beta-1, beta-2, beta-3, tau-1). Unfortunately, beta-1 had better things to do and died away, rest in peace. The alpha and beta colonies grew on two plates that contained sample B (48 ul Accepting Vector, 16 ul Insert) while the tau colony grew on a plate that contained sample A (2 ul Accepting Vector, 4 ul Insert). Only one colony grew from sample A (tau-1) while 15-20 colonies grew from sample B. The nanodrop results above indicate a very high concentration of pNIC-Bsa4 with Insert, showing promise for yield considerations. Additionally, the 260/230 and 260/280 absorbance values are in the recommended range, though higher 260/230 values would be more optimistic.
Another note: During the transformation step, 15 minutes passed by before SOC media was added after 30 minutes in ice. Additionally, the time in the shaking incubator was cut to 40 minutes.
September 11, 2012 Accepting vector pNIC-Bsa4 digest verification with Gel Electrophoresis
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: pNIC-Bsa4 cut with BsaI (AB)
Lane 4: Skip
Lane 5-6: pNIC-Bsa4 cut with BsaI (KR)
Lane 7-10: Skip
PCR Clean Up pNIC-Bsa4
260/280 ratio is 1.85, 260/230 ratio is 2.29. Concentration is 49.8 ng/ul. Sample is usable as an accepting vector.
260/280 ratio is 1.90, 260/230 ratio is 2.34. Concentration is 68.1 ng/ul. Sample is usable as an accepting vector.
Week 1
September 07, 2012 PCR^2 cleanup Nanodrop
Absorbance ratios are 1.89 at 260/280 (indicative of pure DNA) and 1.99 at 260/230 (another indication of pure DNA). Concentration is 105.0 ng/ul, suitable for inserting vector preparation and bacteria transformation.
Gel check of all PCR samples to determine viability.
Lane 1: Primary alpha - PCR done on 09.06.12
Lane 2: Primary gamma - PCR done on 09.06.12
Lane 3: Primary alpha - PCR done on 09.04.12
Lane 4: Primary beta - PCR done on 09.04.12
Lane 5: Primary gamma - PCR done on 09.04.12
Lane 6: Primary Sample A
Lane 7: Primary Sample C
Lane 8: Secondary gamma - PCR done on 09.06.12
Lane 9: Secondary from 08/01 sample
Lane 10: Secondary from 07/18 sample
All samples seem to be okay, other than Lane 9 DNA sample. September 06, 2012
Gel check of secondary PCR^2 from September 04, 2012 - unsuccessful. Samples were left at 4 degrees Celsius.
Tested Q5 High Fidelity Hot Start Polymerase with recommended NEB guidelines for primary PCR (20 cycles) - successful.
Secondary PCR with Q5 HF was done (KOD thermocycling protocol) - successful.
Lane 1: skip
Lane 2: 1 kb DNA Ladder
Lane 3-6: secondary PCR^2 (5 ul) KOD protocol
Lane 7-8: skip
Lane 9: ADO primary
Lane 10: ADO secondary
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: MM primary PCR
Lane 4: MM secondary PCR
Lane 5: KR primary PCR (alpha)
Lane 6: KR primary PCR (beta)
Lane 7: KR primary PCR (gamma)
Lane 8: skip
Lane 9: skip
Lane 10: skip
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: PCR^2 Sample ?
Lane 4: PCR^2 Sample ?
Lane 5-10: Skip
September 04, 2012
Tested Q5 High Fidelity Hot Start Polymerase with KOD thermocycling protocol. Primary (30 cycles) and Secondary PCR were done.
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: Primary PCR alpha (06/18/12) (5 ul)
Lane 4: Primary PCR beta (07/03/12) (5 ul)
Lane 5: Primary PCR gamma (08/01/12) (5 ul)
Lane 6-10: Skip
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: Secondary PCR alpha (06/18/12) (5 ul)
Lane 4: Secondary PCR beta (07/03/12) (5 ul)
Lane 5: Secondary PCR gamma (08/01/12) (5 ul)
Lane 6-10: Skip
Both overlap extension PCR worked, indicating that the KOD protocol (slightly modified with higher denaturation temp. at 98 Centigrade, and higher extension temp. at 72 Centigrade. Lane 3 in the Secondary PCR trial failed, most likely to negligence when some sample was lost before thermocycling. August 29, 2012
Tested Q5 High Fidelity Polymerase (No hot start) with thermocycle protocol given by the info. booklet. August 02, 2012
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Alpha primary PCR
Lane 4: Alpha secondary PCR
Lane 5: Beta primary PCR
Lane 6: Beta secondary PCR
Lane 7: Gamma primary PCR
Lane 8: Gamma secondary PCR
Gel results above indicate poor amplification of DNA. Other researchers experienced poor results also, indicating the KOD polymerase was not of usable quality anymore.
August 01, 2012
All 12 samples did not contain DXR recombinant gene. Only tail-end primers were sequenced, indicating contamination by smaller DNA fragments.
July 30, 2012
Submitted 12 samples from July 27, 2012 to DNA Sequencing
July 27, 2012
Restriction Enzyme PvuII Digest of Miniprep Samples
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Control pNIC-Bsa4 No Insert
Lane 4: pNIC-Bsa4 A3 No.1 Insert
Lane 5: pNIC-Bsa4 A4 No.2 Insert
Lane 6: pNIC-Bsa4 A4 No.3 Insert
Lane 7: pNIC-Bsa4 B4 No.4 Insert
Lane 8: pNIC-Bsa4 A3 No.5 Insert
Lane 9: pNIC-Bsa4 B4 No.6 Insert
Lane 10: Skip
Star Activity is visible in Lanes 9&10.
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Control pNIC-Bsa4 No Insert
Lane 4: pNIC-Bsa4 A4 No.7 Insert
Lane 5: pNIC-Bsa4 A4 No.8 Insert
Lane 6: pNIC-Bsa4 A3 No.9 Insert
Lane 7: pNIC-Bsa4 B3 No.10 Insert
Lane 8: pNIC-Bsa4 B3 No.11 Insert
Lane 9: pNIC-Bsa4 A3 No.12 Insert
Lane 10: Skip
Star Activity is visible in Lanes 6 thru 9.
Incubated for 3 hours at 37 degrees Celsius. No heat-block treatment past the water-block. This may explain the star activity in several of the lanes above.
July 25, 2012
Made master-plate and 12 tubes to incubate for 16 hours.
July 24, 2012
Prepared 15*1ml aliquots 1 mg/ml Kanamycin Stock. Few colonies are visible on the four plates grown yesterday (July 23, 2012)
July 23, 2012
Annealed and Transformed PCR product 3 (214) and PCR product 4 (170) with newly cut pNIC-Bsa4 Vector (?) and AH pNIC-Bsa4 Vector.
July 20, 2012
Restriction Enzyme Digest, Verification of PCR Product and Accepting Vector, More pNIC-Bsa4 cut
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: PCR^2 AH
Lane 4: 1A Miniprep Sample July 18, 2012
Lane 5: 1B Miniprep Sample July 18, 2012
The stream of DNA in Lane 4 and Lane 5 indicate that we have ended up with extraneous DNA.
To prepare for another trial run, we analyzed newly made inserting PCR product and accepting vector.
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: PCR Clean Up 3?
Lane 4: PCR Clean up 4?
Lane 5: 072012 pNIC-Bsa4 Cut Vector
Lane 6: 1A Miniprep Sample July 18, 2012
Lane 7: 1B Miniprep Sample July 18, 2012
pNIC-Bsa4 cut (started at 133 ng/ul) concentration, 27.1 ng/ul July 19, 2012
Annealing and Transformation Take 2, PCR strikes back
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: AB pNIC Cut
Lane 4: AB pNIC Cut
Lane 5: Skip
Lane 6: KR Secondary PCR
Nanodrop concentration: 213.9 ng/ul (eluted in 30 ul 10mM Tris HCl)
Nanodrop concentration: 170.2 ng/ul (eluted in 30 ul 10 mM Tris HCl)
We also submitted PCR Insert and pNIC-Bsa4 accepting vector (July 16, 2012 and July 17, 2012) after cohesive end generation to DNA sequencing to determine whether cohesive ends had been made before annealing and transformation. July 18, 2012
PCR^2 Clean Up, Gel Analysis of Cut vector and PCR Insert (before and after cohesive ends).
Nanodrop P.fal after PCR Clean Up 1A - Concentration 76.8 ng/ul, Absorbance ratios indicate pure DNA. Contaminated
Nanodrop P.fal after PCR Clean Up 5B - Concentration 71.5 ng/ul, Absorbance ratios indicate pure DNA. Contaminated
July 27, 2012. The samples above did not contain the insert. The absorbance spectrums most likely indicate the presence of chromosomal DNA.
Lane 1: Skip
Lane 2: AB pNIC-Bsa4 Cut
Lane 3: AB pNIC-Bsa4 Cut
Lane 4: Skip
Lane 5: PCR Clean Up 3? KR
Lane 6: PCR Clean Up 4? KR
Lane 7: pNIC-Bsa4 Cut (after cohesive end generation)
Lane 8: PCR insert (after cohesive end generation)
Gel above revealed that PCR product and accepting vector were 'usable' - but does not demonstrate if cohesive ends had been formed.
July 17, 2012
Re Digest No. 2 with PvuII and Cohesive End Gel analysis
Lane 1: 1 kb DNA Ladder
Lane 2: AB pNIC Cut
Lane 3: pNIC-Bsa4 (Uncut, no Insert)
Lane 4: pNIC-Bsa4 (Cut with PvuII, no Insert)
Lane 5-9: pNIC-Bsa4 (Cut with PvuII, with insert)
We ran the restriction digest again to verify the findings from July 16, 2012. The results from DNA sequencing indicate no matches with the gene of interest. July 16, 2012
RE Digest with PvuII and DNA Sequencing
Lane 1: skip
Lane 2: 1kb DNA Ladder
Lane 3: pNIC-Bsa4 (no PvuII cut, no insert)
Lane 4: pNIC-Bsa4 (after PvuII cut, no insert)
Lane 5-9: pNIC-Bsa4 (after PvuII cut, with insert)
Lane 10: skip
Unexpectedly, there are no bands in Lanes 5-9. This indicates that vector with the gene insert had not been transformed, even though we witnessed colony growth on the master plate.
Samples were submitted for DNA sequencing. July 13, 2012
Miniprep to extract and purify plasmid DNA from tubes
2 ul Sample 2 at 260 nm. Contaminated
2 ul Sample 2 Trial 2 at 260 nm. Contaminated
2 ul Sample 4 at 260 nm. Contaminated
2 ul Sample 4 Trial 2 at 260 nm. Contaminated
2 ul Sample 6 at 260 nm. Contaminated
2 ul Sample 6 Trial 2 at 260 nm. Contaminated
2 ul Sample 7 at 260 nm. Contaminated
2 ul Sample 7 Trial 2 at 260 nm. Contaminated
2 ul Sample 8 at 260 nm. Contaminated
2 ul Sample 8 Trial 2 at 260 nm. Contaminated
Only colonies in tubes 2,4,6,7,8 produced a pellet after we centrifuged the conical tubes and removed supernatant. This is surprising since all tubes were incubated in similar conditions. We may have chosen 'bad' colonies (colonies were too large) in tubes 1,3,5. After plasmid preparation, the plasmid was eluted in 50ul Elution Buffer. The nanodrop results had concentrations between 25 ng plasmid/ul to 120 ng plasmid/ul. July 27, 2012. None of the samples contained the insert. The high 260/230 absorbance ratios may indicate that samples have been contaminated. The samples in this trial were chosen from plates that contained many ( ~100 to 200 colonies), indicating that the kan. did not kill cells that did not transform. July 12, 2012
Protein Purification of FtHAP, Master Plate Growth of P.fal. Dxfr
Elution 1 FtHAP
Elution 2 FtHAP
The nano drop measurements reveal very low absorbance values of protein. This is not surprising, as we deviated from protocol often.
8 colonies from the 2 plates transformed with the pNIC-Bsa4 vector were selected to grow in 8 tubes and one agar plate at 37 degrees Celsius.
July 11, 2012
Gel Check of pNIC-Bsa4 cut with BsaI after PCR clean-up
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: 2.5 ul pNIC-Bsa4 digested with BsaI+ 0.5 ul blue dye
Lane 4-10: Skip
The two bands indicate two cuts were made, indicative of good result. Consequently, we made cohesive ends for the accepting vector (pNIC-Bsa4) and the inserting PCR DNA product (P.fal gene). The two pieces were annealed and transformed in 25 ul of DH5a Competent Cells and plated (2 plates) for overnight incubation. July 10, 2012
Nanodrop pNIC-Bsa4
We cut the plasmid with the Bas1 Restriction Enzyme for 3 hours at 37 degrees Celsius. After PCR-Clean Up, we measured the concentration of plasmid - 40.8 ng/ul. July 09, 2012
Nanodrop P.fal. DXFR (Sample A)
We did PCR Clean Up using the PCR^2 aliquots and measured the concentration of the DNA from scratch using the spectrophotometer. The average concentration (n=2) is 71.25 ng/ul. July 06, 2012
Secondary PCR and PCR^2
Lane 1: Skip Lane 1: Skip
Lane 2: 1kb DNA Ladder Lane 2: 1kb DNA Ladder
Lane 3: PCR^2 Aliquot 1 A Lane 3: PCR^2 Aliquot 1 C
Lane 4: PCR^2 Aliquot 2 A Lane 4: PCR^2 Aliquot 2 C
Lane 5: PCR^2 Aliquot 3 A Lane 5: PCR^2 Aliquot 3 C
Lane 6: PCR^2 Aliquot 4 A Lane 6: PCR^2 Aliquot 4 C
Lane 7: Skip Lane 7-10: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Secondary PCR A
Lane 4: Secondary PCR C
Lane 5: Secondary PCR C (Sample tube was crushed in the thermocycler, also prematurely stopped final elongation).
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Both Secondary PCR and PCR^2 have worked. July 05, 2012
Primary and Secondary PCR
Lane 1: Skip
Lane 2: 100 bp DNA Ladder
Lane 3: 1kb DNA Ladder
Lane 4: Primary PCR (PA)
Lane 5: Secondary PCR Option B (PA)
Lane 6: Skip
Lane 7: Primary PCR, old Oligo Mix A (KR)
Lane 8: Primary PCR, new Oligo Mix B (KR)
Lane 9: Primary PCR, new Oligo Mix C (KR)
Lane 10: Skip
(Continuing from July 02, 2012)
Comparing Lane 8 and Lane 9, Oligo Mix B was not properly prepared. Consequently, Oligo Mix B was discarded for future tests. The similarity in DNA product in Lane 7 and Lane 9 suggests that the old Oligo Mix was correctly prepared, but mistakes were made while preparing Secondary PCR. July 03, 2012
Remade Oligo-Mix (two separate batches). July 02, 2012
Lane 1: Skip
Lane 2: 1kb DNA Ladder, 5 ul
Lane 3: Secondary PCR Option B, 15 ul
Lane 4: Skip
Lane 5: Skip
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
(Continuing from June 29, 2012)
The amplified Gene of Interest DNA is faintly visible in Lane 3. From the line matching up with the 1.5k bp, the band dims. Even though the band should be intense, it may be enough to validate the visible band from June 29, 2012. June 29, 2012 Looks good - hopefully the next round turns out better. - Dr. B
Lane 1: Skip
Lane 2: Skip
Lane 3: 1kb DNA Ladder
Lane 4: Primary PCR or Secondary PCR Option B? [KR]
Lane 5: Primary PCR or Secondary PCR Option B? [KR]
Lane 6: Skip
Lane 7: Skip
Lane 8: 100 bp DNA Ladder [UM]
Lane 9: primary PCR [UM]
Lane 10: secondary PCR B [UM]
(Continuing from June 27, 2012)
In this attempt to clone the gene with custom primers, we changed the annealing temperature to 59 degrees celsius, diluted the custom primers in 10 ul final volume (previously made 5 ul final volume), and stored the PCR product in the -20 fridge immediately after the PCR cycling finished. However, the samples did not fluoresce well, evident above. Assuming the band in lane 3 is the 3kb DNA ladder fragment, then the band in lane 4 may be 1500 bp, which is the size of the gene we are studying.
We also monitored growth and harvesting of competent BL21 cells for protein expression.
June 28, 2012
Made 10x 50 ml and 5x 500 ml LB media for growing bacteria. June 27, 2012
Lane 1: 1 kb DNA Ladder
Lane 2: primary PCR [AH]
Lane 3: secondary PCR [AH]
Lane 4: secondary PCR B new (but old custom primers) [KR]
Lane 5: secondary PCR B new (but new custom primers) [KR]
Lane 6: secondary PCR A new (but old Oligo Mix primers) [KR]
Lane 7: secondary PCR A new (but new Oligo Mix primers) [KR}
Lane 8: secondary PCR A old [KR]
Lane 9: primary PCR new [KR]
Lane 10: secondary PCR B old [KR]
(Continuing from June 26, 2012)
None of the PCR products fluoresced under UV. The faint stream in Lane 9 indicates that Primary PCR (with a newer sample of custom oligonucleotides) may have worked, but it is pale in comparison to Alex H. Primary PCR streak in Lane 2, indicating that it is not accurate. June 26, 2012
Secondary PCR Overlap Option B
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: Skip
Lane 4: Secondary PCR (Option B) KR
Lane 5: Skip
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
(Continuing from June 22, 2012)
Despite increasing the annealing temperature to 65 degrees Celsius, there is no resultant amplified DNA (would have been visible in Lane 4). This indicates that the product was not amplified. Either the template (primary PCR product) or the custom primers may be erroneous. (June 27, 2012) Annealing temperature is not 65 degrees Celsius; should be 60 degrees Celsius. See June 22, 2012. June 25, 2012
PCR pNIC-Bsa4
Primers: pLIC-for and pLIC-rev
Lane 1: Skip
Lane 2: 100 bp DNA ladder 5 ul
Lane 3: ? ng pNIC-Bsa4 KRAB
Lane 4: ? ng pNIC-Bsa4 KRAB
Lane 5: Control KRAB
Lane 6: Skip
Lane 7: ? ng pNIC-Bsa4 AH
Lane 8: ? ng pNIC-Bsa4 AH
Lane 9: ? ng pNIC-Bsa4 AH
Lane 10: Control AH
Sample No. 3 KRAB (smallest amount of pNIC-Bsa4) is not shown above because it was misplaced after being taken out of the PCR machine.
June 22, 2012
Secondary PCR Overlap Option B
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: 100 bp DNA Ladder
Lane 4: Primary PCR AB
Lane 5: Secondary PCR (Option A) AB
Lane 6: Secondary PCR (Option B) AB
Lane 7: Secondary PCR (Option B) KR
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Sample (prepared on June 21, 2012) was stored overnight at 4 degrees celsius. After electrophoresis the gel didn't want to get analyzed, so it slipped onto the ground. We then used 15 ul of the same sample on another gel, above. However, the DNA in this Lane 7 forgot to fluoresce. The annealing temperature may be suspect. We did not change the binding temperature from the Primary PCR. Since the melting temperature of the pNIC-Bsa4 custom primers is approximately 70 degrees Celsius, an annealing temperature of 65 degrees Celsius may yield better results. (June 27, 2012) The previous statement is incorrect. We added 3 ul of 25 mM MgSO4 into 50 ul total volume, resulting in 1.5 mM MgSO4. The melting temperature of the custom primers is about 65 degrees Celsius at this concentration of Mg. The correct annealing temperature is around 60 degrees Celsius.
062112- Kaarthik. Nice job. Can you add a caption specifying the ladder size, your predicted gene size, and what the two different lanes are.
Thanks, Dr. B June 20, 2012
Primary PCR Overlap and Secondary PCR Overlap Option A
Lane 1: Skip
Lane 2: Skip
Lane 3: 1 kb DNA Ladder
Lane 4: Primary PCR AB
Lane 5: Secondary PCR (Option A) AB
Lane 6: Primary PCR KR
Lane 7: Secondary PCR (Option A) KR (Approx. 1.5 kb).
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
The smear from the primary PCR is visible on Lane 6 and the secondary PCR product band is visible on Lane 7. It has a size of 1.5 kb. The actual gene size is 1467 bp. June 20, 2012
PCR pNIC-Bsa4
Primers: pLIC-for and pLIC-rev
Lane 1: 100 bp DNA ladder 5 ul
Lane 2: 0.00824 ng pNIC-Bsa4 DB
Lane 3: 0.0824 ng pNIC-Bsa4 DB
Lane 4: 0.824 ng pNIC-Bsa4 DB
Lane 5: 0.00824 ng pNIC-Bsa4 KR
Lane 6: 0.0824 ng pNIC-Bsa4 KR
Lane 7: 0.824 ng pNIC-Bsa4 KR
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
We used an annealing temperature of 48 degrees Celsius. June 19, 2012
Taking a break. June 18, 2012
Made Oglio-Mix. June 15, 2012
Nanodrop pGBR22 June 15, 2012
PCR pmCherry using VDS Primers and M13 Primers
Lane 1: Skip
Lane 2: 100 bp DNA ladder 5 ul
Lane 3: 0.018 ng pmCherry VDS Set
Lane 4: 0.18 ng pmCherry VDS Set
Lane 5: 1.8 ng pmCherry VDS Set
Lane 6: Skip
Lane 7: 0.018 ng pmCherry M13 Set
Lane 8: 0.18 ng pmCherry M13 Set
Lane 9: 1.8 ng pmCherry M13 Set
Lane 10: Skip
There should be a dark distinct band instead of a faint band since Lane 9 contained 1.8 ng pmCherry. Maybe it jumped over to Lane 10. June 14, 2012
Primer Design
We designed a forward and reverse primer that will allow us to PCR the P.fal. gene and insert it into an expression vector (pNIC-Bsa4).
Forward Primer: 5’ TACTTCCAATCCATGAAAAAGTATATCTACATCTAC 3’ 36 bp GC Content 30.6% 0 mM Mg2+ Tm 56.7 oC 1.5 mM Mg2+ Tm 64.9 oC 2 mM Mg2+ Tm 65.4 oC 4 mM Mg2+ Tm 66.5 oC 6 mM Mg2+ Tm 67.1 oC
Reverse Primer: 5’ AAACACAACTCTTCTTGA 3’ Reverse complement it: 5’ TATCCACCTTTACTGTCAAGAAGAGTTGTGTTT3’ 33 bp GC Content 36.4% 0 mM Mg2+ Tm 59.7 oC 1.5 mM Mg2+ Tm 67.6 oC 2 mM Mg2+ Tm 68.1 oC 4 mM Mg2+ Tm 69.2 oC 6 mM Mg2+ Tm 69.7 oC
June 13, 2012
PCR of pGBR22 using M13 Primers
We repeated the experiment from June 07, 2012 but used the M13 Primer Set instead of the SP6/T7 Primer Set.
Lane 1: Skip
Lane 2: 100bp DNA ladder
Lane 3: 0.3 ng (1% conc.) pGBR22 plasmid KR
Lane 4: 3 ng (1% conc.) pGBR22 plasmid KR
Lane 5: 30 ng (10% conc.) pGBR22 plasmid KR
Lane 6: Control. No pGBR22 plasmid
Lane 7: 0.3 ng (1% conc.) pGBR22 plasmid AB
Lane 8: 3 ng (1% conc.) pGBR22 plasmid AB
Lane 9: 30 ng (10% conc.) pGBR22 plasmid AB
Lane 10: Control. No pGBR22 plasmid. Band maybe contamination from Lane 9. June 11, 2012
Transformation of DH5alpha cells with pGFP
NOTE: PLATE A AND C WERE REVERSED
Plate A: 25ng of Plasmid, 224 colonies/ng
Plate B: 5ng of Plasmid, 800 colonies/ng
Plate C: 1ng of Plasmid, 1200 colonies/ng
June 07, 2012
Amplify Purple Protein coding sequence in the pGBR22 plasmid using Forward and Reverse Primers
PCR of pGBR22 using SP6/T7 Primers
Lane 1: Skip
Lane 2: 100bp DNA ladder
Lanes 3-8: PCR Samples of pGBR22 using SP6/T7 Primers. No fragment bands are visible.
We may have erred in pipetting or stoichiometric calculations. One plausible reason for the absence of any DNA fragments may be incorrect thermocycling programming (the annealing temperature entered corresponded to the M13 primer set, not the SP6/T7 primer set). June 07, 2012
Restriction Digest Result Gel:
Lane 1: Skipped
Lane 2: DNA Ladder
Lane 3: Uncut Plasmid AB
Lane 4: EcoRI AB
Lane 5: PvuII AB
Lane 6: EcoR1+ PvuII AB
Lane 7: EcoRI KR
Lane 8: PvuII KR
Lane 9: EcoR1+ PvuII KR
Lane 10: Uncut Plasmid KR
~1.8 is generally accepted as “pure” for DNA
260/230 Ratios: measure of nucleic acid purity...often higher than the respective 260/280 values.
~2.0-2.2.
PfDXR extinction coefficient: ~39162.5 M-1 cm-1
PfDXR molecular weight: 47206.6 Da
Screened Libraries (DONE)
HF9_180_Plates_1um3D_catnum.sdf
HF9PlatesPlates5_9.sdf
cb_306_3d.sdf
NIH_ClinCol3Ded
ChemBridge-3D
Fragment-set_3D
MayBridge50k_3D
MW-set_3D
Useful Libraries (Docking programs indicate compounds may be of use in these libraries)
cb_306_3D.sdf
ChemBridge-3D
MayBridge50k_3D
MW-set_3D
HF9_Plates_1um3D_catnum.sdf
Screened Libraries (TO DO)
chembIntd (Skip - paper indicates compounds do not target DXR)
MAR3D7 (Skip - paper indicates compounds do not target DXR)
Microsets
January 21, 2013
PreFPLC PfDXR concentrated to 0.5 ml - takes about 50 min
yield: 3.39 mg
Used Umeda Buffers (all include 2 mM DTT - reducing agent) for protein purification
Also learned what happens when the pH drops below 7 in protein solutions and when you fail to remove the sticker from the bottom the SDS Page Gel.
Week 13
Freebie from Journal Club Team B victory
Week 12
November 30, 2012
Enzymatic Assay not promising
Several enzyme assays (varying in room temp. vs 37 degrees Celsius and varying in duration from 5-15 min) were performed. Both snap frozen and -20 stocks of PfDXR were used. Absorbance of NADPH was measured at 340 nm. All trials revealed a constant gradient for NAPDH absorbance, indicating that NADPH was not being oxidized.
The obvious conclusion is that the PfDXR enzyme we synthesized is not functional. However, purification, characterization and concentration were all completed within a span of 12 hours, so the probability the enzyme denatured is low.
An alternative is to modify the assay parameters. Based on literature searches, the optimum temp. for PfDXR activity is 37 degrees Celsius. Most of the trials were done at room temp., which may explain why the absorbance was flat lined. For the two assays done at 37 degrees Celsius, one was done for 5 min. only while the other was "oversaturated" with enzyme - therefore, changing the optimal conc. of buffer and reagents. (NEED to verify the last sentence with data on computer in lab).
The remaining option is to redo the assay at 37 degrees Celsius with the following changes.
1. 5 min incubation with reagents and buffer at 37 degrees Celsius in PCR thermocycler.
2. Add PfDXR enzyme and obtain NADPH absorbance at 340 nm.
3. Incubate at 37 degrees Celsius for 10-20 min.
4. Read the absorbance value at 340 nm again.
If the above steps do not reveal a depletion of NADPH (i.e. 0 absorbance at 340 nm after 10-20 min), then all hope is lost - the enzyme is inactive.
November 28, 2012
Prepared enzyme assay reagents
NADPH (cofactor) and DXP (substrate) were solubilized in 0.01 M NaOH and pure water, respectively to obtain 192 5 mM NADPH aliquots (5 enzyme assays each) and 175 12 mM DXP aliquots (1 enzyme assay each). Tris-HCl was diluted to 125 mM.
November 27, 2012
Protein sonication (lysing), purification (extraction), characterization (verification), and concentration
PfDXR concentrated pre-FPLC (1 ml)
A = 0.235
e = 39162.5 M-1 cm-1
b = 1 cm
c = A/Eb = (0.235) / (39162.5 M-1 cm-1) * (1 cm) = 6 microMolar.
6 microMolar = (6 micro moles / 1 liter) * (47206.6 g/mole) = 0.283 mg/ml
yield = 0.283 mg
PfDXR homodimer is present in fraction samples 25 through 29. Presence can be verified by molecular standards (77 kDa is the small bump near fraction samples 29 through 31). PfDXR monomer is approx. 47 kDa (dimer will be approx. 94 kDa).
Rescaled FPLC graph (same as above). Small bump at fractions 31 through 34 may indicate monomer - standard at fractions corresponding to 33 through 36 is approx. 33 kDa.
PfDXR concentrated post-FPLC in glycerol (0.5 ml)
A = 0.291
e = 39162.5 M-1 cm-1
b = 1 cm
c = A/Eb = (0.291) / (39162.5 M-1 cm-1) * (1 cm) = 7.43 microMolar.
7.43 microMolar = (7.43 micro moles / 1 liter) * (47206.6 g/mole) = 0.351 mg/ml
yield: 0.1755 mg
PfDXR concentrated post-FPLC snap frozen (0.5 ml)
A = 0.294
e = 39162.5 M-1 cm-1
b = 1 cm
c = A/Eb = (0.294) / (39162.5 M-1 cm-1) * (1 cm) = 7.51 microMolar.
7.51 microMolar = (7.51 micro moles / 1 liter) * (47206.6 g/mole) = 0.354 mg/ml
yield: 0.177 mg
total yield after FPLC: 0.354 mg
November 26, 2012
IPTG induction, 20 hour overnight expression
112612 - ??. Dr B
Week 11
November 21, 2012
Ranking of Libraries by top scoring ligands (11/21/12)
1. ChemBridge-3D
2. HF9_180_Plates_1um3D_catnum
3. MayBridge50k_3D
4. MW-set_3D
5. cb_306_3d
6. NIH_ClinCol3Ded
7. Fragment-set_3D
8. HF9PlatesPlates5_9
Top Ten Ligand List did not change from Week 8 results. However, MayBridge50k_3D entered the top 20 list and MW-set_3D entered the top 30 list.
Top Compound No. 1 7576671 docked in active site of PfDXR
7576671 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
Top Compound No. 3 7969923 docked in active site of PfDXR
7969923 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
Top Compound No. 4 5583722 docked in active site of PfDXR
5583722 shown as sticks (Carbon purple). MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
FOM docked in active site of PfDXR
FOM shown as sticks (Carbon purple).
MG ion shown as sphere (Orange). Active site residues within 5 A shown as sticks (Carbon light blue). Hydrophobic residues shown as lines (Carbon green)
November 20, 2012
We went ahead and transformed colonies 5 and 15 into BL21 (DE3) competent cells. Also, we started a 320mL culture of colony 5 for Midiprep. Will spin down and Midiprep tomorrow.
November 19, 2012
DNA sequencing shows positive clones for submitted samples
Miniprepped plasmid samples from colonies 5,6,15,16, 21 & 22. A BLAST comparison indicated positive clones for every colony without mutations.
Week 10 -
good - Dr. B 11/19/12
November 18, 2012
RE Digest with HincII enzyme to check for positive clones
Virtual Gel of PfDXRL75 - positive clones on gel image should have bands at sizes indicated above.
Gel Image of RE Digest of PfDXR in pNIC-Bsa4
with HincII for Colonies 1-8
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: Colony 1 - not likely to be full insert
Lane 4: Colony 2 - not likely to be full insert
Lane 5: Colony 3 - not likely to be full insert
Lane 6: Colony 4 - not likely to be full insert
Lane 7: Colony 5 - positive clone (sequenced)
Lane 8: Colony 6 - positive clone (sequenced)
Lane 9: Colony 7 - likely a positive clone (did not sequence)
Lane 10: Colony 8 - likely a positive clone (did not sequence)
Gel Image of RE Digest of PfDXR in pNIC-Bsa4
with HincII for Colonies 9-16
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: Colony 9 - likely a positive clone (did not sequence)
Lane 4: Colony 10 - likely a positive clone (did not sequence)
Lane 5: Colony 11 - likely a positive clone (did not sequence)
Lane 6: Colony 12 - likely a positive clone (did not sequence)
Lane 7: Colony 13 - not likely to be full insert
Lane 8: Colony 14 - not likely to be full insert
Lane 9: Colony 15 - positive clone (sequenced)
Lane 10: Colony 16 - positive clone (sequenced)
Gel Image of RE Digest of PfDXR in pNIC-Bsa4
with HincII for Colonies 17-24
Lane 1: Skip
Lane 2: 1 kb Ladder
Lane 3: Colony 17 - likely a positive clone (did not sequence)
Lane 4: Colony 18 - likely a positive clone (did not sequence)
Lane 5: Colony 19 - likely a positive clone (did not sequence)
Lane 6: Colony 20 - not likely to be a positive clone
Lane 7: Colony 21 - positive clone (sequenced)
Lane 8: Colony 22 - positive clone (sequenced)
Lane 9: Colony 23 - not likely to be a positive clone
Lane 10: Colony 24 - not likely to be a positive clone
November 17, 2012
Master Plate Protocol completed on 24 colonies
November 16, 2012
Cut pNIC and Cloning and Transformation
cut pNIC-Bsa4 at BsaI sites - combined two samples (starting conc. 82.4 ng/ul)
November 15, 2012
More PCR from plasmid
combined 5 samples (20 cycles each)
November 14, 2012
Back to cloning part 2
Transformation was not successful
November 13, 2012
Back to cloning
We used the new L75 forward primer to do PCR.
Lane 1 & 5-10: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: 10 ul PfDXRL75 (~1300 bp) - used 1 ng in 50 ul reaction volume, 20 cycles
Lane 4: 10 ul PfDXRL75 (~1300 bp) - used 5 ng in 50 ul reaction volume, 20 cycles
PCR Clean Up was not as fruitful (21.9 ng/ul), but satisfactory
Week 9
November 08, 2012
PCR from primary mix- used wrong forward primer
supersec: 20 sec annealing
kr2: 1 min annealing
Lane 3: 1A primary smear -primary mix made in June kr2 protocol
Lane 4: 1B primary smear -primary mix made in July kr2 protocol
Lane 3: 1A primary smear - primary mix made in June supersec protocol
Lane 4: 1B primary smear - primary mix made in July supersec protocol
Lane 5: 2A secondary - kr2 protocol
Lane 6: 2B secondary - kr2 protocol
secondary from 1B
Lane 1&6: 100 bp Ladder
Lane 2: 2B secondary - kr2 protocol
Lane 3: 2B secondary - kr2 protocol
Lane 4: 2B secondary - supersec protocol
Lane 5: 2B secondary - supersec protocol
PCR^2 from 2B secondary -kr2 protocol
November 07, 2012
Combined efforts no go - used wrong forward primer
Andrew, Alex and I gave it another attempt at PCR from the plasmid DNA.
Lane 1: 1kB DNA Ladder
Lane 2-5: Alex's vain attempt using modified Aptamer PCR protocol on plasmid DNA (15 cycles)
Lane 6: Kaarthik's vain attempt using custom protocol on plasmid DNA (30 cycles)
Lane 7: Kaarthik's vain attempt using primary mix DNA with custom protocol. (30 cycles)
Lane 8: Andrew's vain attempt using Aptamer protocol (no modifications) on plasmid DNA (15 cycles)
Lane 9-12: Recheck of secondary PCR with new primers (20 cycles only).
Looks like we made a new DNA ladder!
We will remake the primary DNA and use the new forward to make secondary PCR DNA.
November 04 and 05 and 06, 2012
PCR Party no go - used wrong forward primer
PCR on the plasmid DNA was attempted several times to no avail. I went back to the primary 1A mix (first mix made in the June 2012) and used it as template with the new forward primer. The secondary DNA output appeared to be the correct size in bp, but PCR^2 from this secondary resulted in smaller amplicon length.
After these successive failures, I will do a dpnI digest of the 17 ng/ul DNA and anneal/transform. The only downside is that the gene appears to be near 1500 bp (suggesting I used the wrong forward primer) but the gel can be deceiving.
Lane 1: 1kB DNA Ladder
Lane 2-5: Secondary PCR^2 - wrong gene size
Lane 6-12: Secondary PCR^2 - no DNA product
Lane 1: 1kb DNA Ladder
Lane 2: 1A Primer smear
Lane 3-6: Secondary PCR from 1A primary
Lane 7-10: Another attempt at DNA amplification from plasmid DNA
November 02, 2012
PCR from plasmid DNA - used wrong forward primer
No PCR product - will probably need to adjust timing of denaturation and annealing steps
-maybe try diluting stock plasmid directly (add 500 ul Tris HCl to original amount).
-or do three fold dilution from 90 ng/ul to 45 ng/ul to 22.5 ng/ul to 11.25 ng/ul
November 01, 2012
PCR from plasmid DNA. Incorrectly followed right protocol - used wrong forward primer
Lane 1 & 8-10: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: PCR using overlap extension conditions - 84 ng of plasmid DNA (10 ul added to gel)
Lane 4-7: PCR using plasmid DNA conditions
There was no product in lanes 4-7.
Mistakes made:
1. Used MgSO4 instead of MgCl2
2. Used PCR-Overlap thermocycling conditions instead of recommended protocol.
October 31, 2012
PCR^2 from plasmid DNA. Followed wrong protocol - used wrong forward primer
DNA replication results were not promising. We used nearly 84 ng (the KOD info sheet recommends 1 ng only of plasmid DNA) so we need to modify the cycling conditions.
Week 8
Official No Wet Lab Week
No significant work done in lab.
All of the jobs submitted to Maestro for 2D to 3D conversion of the chembIntd and MAR3D7 libraries failed to complete. One job succesfully ran but prematurely exited with a fatal message (FATAL m2io_get_number_blocks(): error getting number of blocks in file) while the other 4 did not begin.
Virtual screening was completed on all of the proposed libraries. Some results follow.
Top Ten Ligands after 2 Runs of Screened Libraries 10/28/12
Ranking of Libraries by top scoring ligands
1. ChemBridge-3D
2. HF9_180_Plates_1um3D_catnum
3. cb_306_3d
4. NIH_ClinCol3Ded
5. Fragment-set_3D
6. HF9PlatesPlates5_9
Top Compound No. 2 RJC00044SC docked in active site of PfDXR
Validation docking was also completed (Autoscale = 2; comparable to above top ten) on known inhibitor Fosmidomycin (FOM)
While FOM has a smaller Score than the top ten compounds, it has the second best S(hbond) and the best S(metal) score.
Original FOM docked in the active site of PfDXR
Original FOM aligned with Validation Docking Pose No. 4 FOM
The Validation indicates that Virtual Screening may provide reliable results for identifying inhibitors.
Week 7
102112- Yeah - keep up the good work. Dr. B
October 19, 2012
Midiprepped PfDXR
Andrew spun down a culture of E. Coli with the recombinant DNA and the DNA was extracted with the Qiagen set.
October 17, 2012
DNA Sequencing Results
Sample 2A5 has the full coding sequence in the pNIC-Bsa4 vector.
Sample 2A5. 77.1 ng/ul.
2A5 Forward:
>lcl|58321
Length=1890
Score = 1844 bits (998), Expect = 0.0
Identities = 1018/1031 (99%), Gaps = 4/1031 (0%)
Strand=Plus/Plus
The Gaps included 2 deletions approximately 100 bp before the start codon which are likely false positives and 2 deletions around the 1050-1060 bp no. range and are false positives since these mutations were not present in the reverse sequence.
2A5 Reverse
>lcl|39031
Length=1890
Score = 1881 bits (1018), Expect = 0.0
Identities = 1030/1042 (99%), Gaps = 0/1042 (0%)
Strand=Plus/Plus
There were no gaps in this read of the target coding sequence. There were several "N" - the actual bp at these locations was verified by reading the peaks displayed in the chromatogram and comparing base location to the forward read.
October 16, 2012
DNA Sequencing Results
The predictions about which samples had the insert were wrong. Neglecting that, there were two positive clones in the sequencing results: 1A2 and 2A3. 1A3 had about 4 deletions and 2A3 had 1 deletion only. There may be more deletions (or other mutations) since we only sent the forward pLIC primer with the samples. Since we know the 2A samples are reliable, we will submit the following samples to sequencing:
2A3 with pLIC-rev, oligoprimer no. 1, oligoprimer no. 38
2A1 with pLIC-for
2A4 with pLIC-for
2A5 with pLIC-for and pLIC-rev
October 15, 2012
DNA Sequencing Submission
The following samples were sent to sequencing:
1A3 1A2 1A5 1A6 1B2 2A3 2A5 2B6
All of them were submitted with the pLIC-for primer.
101612 - Kaarthik - ok good deal. Hopefully the DNA sequencing works. -- Dr. B
Week 6
October 13, 2012
Restriction Enzyme Digest of miniprep samples with HincII
The following restriction enzyme digest indicated the successful transformation of E. Coli DH5a with recombinant DNA pNIC-Bsa4 PfDXR construct, albeit only in a few cases. No DNA ladder was added to the agarose gel because 12 samples were added to each agarose gel (with 12 lanes).
HincII RE Digest of Samples from A & B plates containing 249.2 ng/ul insert.
Lane 1. 1A1 - likely insert
Lane 2. 1A2 - unknown 10/16/12 INSERT: several deletions with pLIC-for
Lane 3. 1A3 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 4. 1A4 - no digest by HincII
Lane 5. 1A5 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 6. 1A6 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 7. 1B1 - likely insert
Lane 8. 1B2 - likely insert 10/16/12 NOT AN INSERT: tail end
Lane 9. 1B3 - no digest by HincII
Lane 10. 1B4 - likely insert
Lane 11. 1B5 - unknown
Lane 12. 1B6 - no digest by HincII
The digest was repeated for the A & B plates containing the 339.1 ng/ul insert
HincII RE Digest of Samples from A & B plates containing 339.2 ng/ul insert
Lane 1. 2A1 - more than likely insert 10/17/12: INSERT: Several deletions in Insert
Lane 2. 2A2 - no digest by HincII
Lane 3. 2A3 - more than likely insert 10/16/12: INSERT: only one deletion present with pLIC-for 10/17/12: Several deletions in Insert
Lane 4. 2A4 - more than likely insert 10/17/12: INSERT: Several deletions in Insert
Lane 5. 2A5 - confirmed insert 10/16/12: INSERT: MAGIC
Lane 6. 2A6 - no digest by HincII
Lane 7. 2B1 - no DNA
Lane 8. 2B2 - no digest by HincII
Lane 9. 2B3 - no digest by HincII
Lane 10. 2B4 - no DNA
Lane 11. 2B5 - no digest by HincII
Lane 12. 2B6 - more than likely insert 10/16/12: NOT AN INSERT: tail end
For the above gel, we cannot conclude if the PfDXR gene is present because a successful cut of the recombinant DNA should produce 5 cuts, with 2 cuts visible and 3 cuts faded - see Sept. 14, 2012 or Sept. 24, 2012).
We made two mistakes in this trial. One, we should have added the DNA Ladder to make comparisons reliable and conclusions easier to validate. Second, we should have not run the gel for 45 minutes - 35 minutes at 110 V may be ideal for a RE Digest, especially since a digest with HincII will produce several DNA strands below 500 bp.
Another conclusion is to decrease the digest time to 1 hour since star activity is visible in both gels above. We will submit the following samples with the pNIC-for primer to DNA Sequencing:
1A3 1A2 1A5 1A6
1B2
2A3 2A5
2B6
October 12, 2012
Miniprep RE Digest
Since there were 24 samples, only measurement (1A1) was uploaded.
The 2B samples may be of low purity because we left ethanol solution in the sample tubes before proceeding to elution the next morning.
Nanodrop 1A1. Concentration 60.7 ng/ul 260/280 - 2.05 & 260/230 - 2.03
October 11, 2012
Master Plate Protocol
Colonies grew overnight on the four plates. We chose 24 colonies (6 from each plate) to continue for miniprep.
October 10, 2012
Cohesive End Generation, Annealing and Transformation
We ran a gel with 1 ul of the 340 ng/ul PfDXR cleaned up and 1 ul of the accepting vector cut with BsaI.
Gel indicating presence of 1.5kB PfDXR gene and cut pNIC-Bsa4
Lane 1: Skip
Lane 2: 1 kB DNA Ladder
Lane 3: PfDXR cleaned up (339.1 ng/ul)
Lane 4: cut pNIC-Bsa4
Lane 5-10: Skip
We also did cohesive end generation with the new T4 polymerase, dGTP and dCTP (purchased in early October). We did the annealing and transformation step verbatim. Four plates were made (No. 1 contained the insert that originally came from the 249.2 concentrated stock and No. 2 contained the insert from the 339.1 concentrated stock) and one set of plates contained 2 ul of accepting vector + 4 ul of insert and the other set of plates contained 48 ul of accepting vector and 16 ul of insert.
October 09, 2012
pNIC-Bsa4 Midiprep, pNIC-Bsa4 vector preparation
We midiprepped pNIC-Bsa4 according to the Qiagen protocol. We doubled the buffer volume (i.e. 6 ul to 12 ul) as was suggested in the booklet to increase DNA yield, but the result of this is uncertain because we ended with a low concentration of the vector - 51.5 ng/ul. The yield may also be low because we did not adhere strictly to time constraints (i.e. incubation times were longer).
Nanodrop of pNIC-Bsa4 eluted in 5 mM Tris HCl. Concentration was 51.5 ng/ul, 260/280 at 1.91, 260/230 at 2.29.
We also cut a 82 ng/ul pNIC-Bsa4 vector with BsaI. The results are promising. In the past, we let the RE Digest go for 2.5 hours, but we increased the time to 3 hours in this instance.
Nanodrop of pNIC-Bsa4 cut with BsaI eluted in 10 mM Tris HCl. Concentration was 71.7 ng/ul, 260/280 at 1.93, 260/230 at 2.30
October 08, 2012
Secondary PCR, PCR^2
We successfully made high concentrations of the PfDXR encoding gene from PCR. We deviated from standard protocol in that we decreased the PCR^2 thermocycling to 25 cycles in the hopes of minimizing mutations.
Verification of Secondary PCR
Lane 1: Skip
Lane 2: 1kB DNA Ladder (3 ul)
Lane 3: Secondary PCR from primary mix C prepared in the beginning of July (5 ul)
Lane 4-10: Skip
We used the KOD polymerase from 09/12/12 - we made sure to immediately transfer the final 50 ul sample to the thermocycler once the KOD was added. This was likely the critical step that we had failed to observe in last week's PCR attempts.
We then repeated the thermocycling with the secondary PCR template from above but did 25 cycles only.
Verification of PCR^2
Lane 1: Skip
Lane 2: 1kB Ladder
Lane 3: Secondary PCR^2 from primary C
Lane 4: Secondary PCR^2 from primary C
Lane 5: Secondary PCR^2 from primary C
Lane 6: Secondary PCR^2 from primary C
Lane 7-10: Skip
Since we hurried, the gel image is poor - we halted 20 minutes prematurely. Fortunately, the 1.5 kB band is visible in Lanes 3,4 and 5. The unevenness of the brightness in these lanes may be attributed to improper mixing of reagents prior to adding KOD polymerase to the master mix.
We then cleaned up the sample and eluted in 10 mM Tris-HCL.
Nanodrop measurement of 2 ul of PfDXR eluted in 50 ul of 10 mM Tris HCl. Concentration at 249.2 ng/ul, 260/280 at 1.89 and 260/230 at 2.42.
The absorbance values are reasonable and the concentration broke a personal record set on July 09, 2012.
We checked the clean up below.
Verification of PfDXR Gene after clean up
Lane 1: Skip
Lane 2: 1kB DNA Ladder
Lane 3: 2 ul of 249.2 ng/ul PfDXR cleaned up
Lane 4-10: Skip
No tail end primers are visible - indicating minimal contamination.
We repeated the thermocycling to produce more PCR^2 to ensure we have enough for cloning. While the absorbance values are in range and the concentration is high, we will need to gel check the sample later to verify the presence of the PfDXR gene.
Nanodrop measurement of 2 ul of PfDXR (Sample No. 2) eluted in 50 ul 10 mM Tris HCL. Concentration at 339.1 ng/ul. 260/280 at 1.85, 260/230 at 2.27.
We will continue with cloning this week.
Week 5
100912 - look great. Keep on truckin' - DR. B
October 05, 2012
Verification of plates, PCR, Gel Check
The VDS Staff plates made in September were okay. At least 50 colonies grew on the plate containing SCP1 in pET. On our transformation attempt, the cohesive end generation may have failed - explaining the lack of colonies on the plates.
We also tried secondary PCR twice - once with the Q5 polymerase and the other with Phusion. Both attempts failed; it is possible that the PCR samples were left on ice too long after the polymerase was added.
Gel Check to verify the presence of DNA in samples
We added 5 ul of sample to each lane
Lane 1: Skip
Lane 2: 1kB DNA Ladder
Lane 3: 1A - Primary prepared in Summer
Lane 4: 1B - Primary prepared in Summer
Lane 5: 1C - Primary prepared in Summer
Lane 6: alpha - Primary prepared in Fall (09/06/12)
Lane 7: gamma - Primary prepared in Fall (09/06/12)
Lane 8: Secondary prepared in Summer
Lane 9: gamma - Secondary prepared in Fall (from the Lane 7 sample)
Next week we will continue with making secondary PCR samples and attempt another transformation.
October 04, 2012
Unsuccessful transformation of pNIC-Bsa4:PfalDXR in DH5-alpha.
No colonies grew on the four LB+Kan+5% Sucrose plates. Other colleagues did not have success on the same batch of plates so we transformed another kanamyacin resistant vector and YopH to determine the efficacy of the plates.
We also prepared another 20 plates with LB+Kan.+5% Sucrose.
October 02, 2012
Annealing and Transformation Take 5, More PCR
The PCR Insert from October 01, 2012 was annealed with the cut pNIC-Bsa4, below. The RE Digest was done over 2.5 hours.
Nanodrop measure of pNIC-Bsa4 digested with BsaI. Concentration 39.1 ng/ul, 260/280 at 2.01 and 260/230 at 2.16.
We also completed several PCR trials. The four PCR^2 samples were done with new dilutions of the forward and reverse primers.
Agarose Gel containing cut pNIC-Bsa4, secondary PCR samples and PCR^2 samples.
Lane 1: 1 kB DNA Ladder
Lane 2: pNIC-Bsa4 Cut after PCR Clean Up
Lane 3: Secondary gamma - original mix from primary 09/04/12 (3 ul) 20 cycles
Lane 4: Secondary alpha - original mix from primary 09/04/12 (3 ul) 20 cycles
Lane 5: Secondary gamma - original mix from primary 09/06/12 (3 ul) 25 cycles
Lane 6: Secondary alpha - original mix from primary 09/06/12 (3 ul) 25 cycles
Lane 7: Skip
Lane 8: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 9: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 10: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 11: PCR^2 - original mix from secondary 07/18/12 (1 ul) 25 cycles
Lane 12 : Skip
We then cleaned up the PCR^2 samples from above. Unfortunately, the concentrations were too low for use and the samples were discarded. We performed two trials and deviated from the default protocol. Instead of combining all four PCR samples into one tube, we combined 2 samples into one tube and continued the clean up from there. As a result, only 100 ul of DNA sample was cleaned up in each trial.
Nanodrop measure of PfDXR; concentration 27.5 ng/ul, 260/280: 1.89, 260/230: 1.29
Nanodrop measure of PfDXR; concentration 29.7 ng/ul, 260/280: 1.99, 260/230: 1.72
October 01, 2012
PCR No Fun
We did another PCR starting from the secondary mix. 4 copies were made, but all were done with 21 cycles only to minimize the possibility of mutations. Decreasing the cycles may have some tradeoffs, including decreased final product yield and increased contamination (more oligoprimers).
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Skip
Lane 4: PCR^2 Sample 1 (3 ul)
Lane 5: Skip
Lane 6: PCR^2 Sample 2 (3 ul)
Lane 7: Skip
Lane 8: PCR^2 Sample 3 (3 ul)
Lane 9: Skip
Lane 10: PCR^2 Sample 4 (3 ul)
Lane 11: Skip
Lane 12: Skip
The difference in band intensity arises from unequal distribution of the master mix to the 4 aliquots. The gel also indicates the presence of a lot of smaller primers (tail end primers) in the PCR samples. This is likely a result of less cycling.
Interestingly, the concentration is comparatively high. The 260/230 absorbance ratio is less then preferred at 1.54
We ran 3 ul of the PCR cleanup Sample on a 1% agarose gel. Surprisingly, there does not seem to be a lot of contamination even though the nanodrop result indicated otherwise.
Lane 1: Skip
Lane 2: 1 kB DNA Ladder
Lane 3-9: Skip
Lane 10: Pfal DXR Clean up (3 ul)
1000112 - Kaarthik - any updates? Dr. B
Week 4
September 28, 2012
DNA Sequencing Analysis for alpha 2, alpha 4, and alpha 12
From 16 colonies, 15 were successfully miniprepped. In the end, 3 colonies had the full coding DNA sequence, albeit with mutations. Most were deletions and are summarized below. The mutations were confirmed with DNA sequencing with five primers, pLIC-for, pLIC-rev, oligonucleotide 1, oligonucleotide 9, and oligonucleotide 38.
alpha 12, at least 7 deletions at bp 423, 424, 524, 1371, 1372, 1374, 1376
alpha 2, at least 7 deletions at bp 423, 424, 524, 1341, 1372, 1374, 1376
alpha 4, at least 2 deletions confirmed at bp 404, 566, one point mutation at bp 898 G replaced for A, another 3 potential mutations at 406, 407, 408 (2 sequencing results indicated deletions at these sites, while one did not).
Since the mutations occur in multiple regions, it is improbable we can do site-directed mutagenesis on these samples. We will continue from secondary PCR but decrease cycling to 20 cycles. This may decrease the number of copies with mutations.
September 24, 2012
Restriction enzyme Digest for 8 more colonies from alpha plate
We ran another digest on the 8 samples from Sept. 21, 2012. There was one positive clone, alpha-12, which was submitted for DNA Sequencing.
Lane 1: Skip
Lane 2-8: alpha n (where n begins at 5 and ends at 11)
Lane 9: alpha 12 pNIC-Bsa4 with Insert cut with HincII
Week 3
September 21 ,2012 - Kaarthik, looks good. Hopefully most of those indels won't turn out to be bad or else we can remove them with site-directed mutagenesis. -- Dr. B
Blast Sequencing Results with custom oligo primers and growth of more colonies from the alpha plate.
The results from sequencing were not positive. A2-for corroborated deletions at 578, 579 and the insertion at 679 while A3-rev corroborated deletions at 578, 579, 679, 1525, 1526, 1528, and 1530. Both A2-for and A2-rev revealed no deletion at 750, however. NOTE: BP deletion sites are wrong. See Sept. 28 analysis for correct data. -- Sept. 28.
The results from the A4 set were more promising but both A4-for and A4-rev indicated deletions at 558, and 720. At 560,561 and 562 there were no deletions and 372 was not sequenced by either DNA sample.
However, in anticipation of unpromising Sequencing results, another 8 colonies were selected from the alpha plate to grow overnight on 9/20. The nanodrop concentrations after miniprep are shown below.
The concentrations are much lower than shown in previous trials with other colonies. The yield may have decreased since the colonies grew over one night, whereas the colonies from Sept. 14 grew over two nights. Another plausible reason is that the centrifuge time was decreased to 5 min (instead of 8 min in the September 14 trials). The next step is to pray that these samples have the complete coding DNA sequence of PfDXR and have a 100% identity match. We will do another restriction enzyme test with HincII before sending (any) positive clones to DNA Sequencing.
September 19, 2012
BLAST Sequencing Results
A2 pLIC-for 98% Max Ident.
A2 pLIC-rev 99% Max Ident.
A4 pLIC-for 98% Max Ident.
A4 pLIC-rev 99% Max Ident.
PfDXR Begins at base pair 155, ends at base pair 1621.
Insertions/Deletions:
A2-for, reliable from 134 to 982: 578 (deletion-C) 579 (deletion-G) 679 (insertion-C). Insertion at 679 not seen in A2-rev.
A2-rev, reliable from 741 to 1673: 750 (deletion-T) 1525 (deletion-C), 1526 (deletion-G), 1528 (deletion-G), 1530 (deletion-A). Deletion at 750 not seen in A2-for.
A4-for, reliable from 78 to 982: 372 (insertion C) 558 (deletion-T) 560 (deletion-C) 561 (deletion-A) 562 (deletion-C) 720 (deletion-C).
Deletion at 720 IS seen in A4-rev.
A4-rev, reliable from 743 to 1683: No mutations.
Will send another sample to DNA Sequencing
A2-for: primer no. 9
A2-rev: primer no. 38
A4-for: primer no. 9
A4-rev: primer no. 38
Kaarthik - nice. - Dr. B 091812
September 17, 2012
DNA Sequencing
Submitted 7 samples to the ICMB Core DSF (500 ng DNA each).
Week 2
September 14 , 2012
Gel Analysis of Restriction Enzyme Digest (HincII) on pNIC-Bsa4 with Insert including controls
Lane 1: 1 kB DNA Ladder (5 ul)
Lane 2: Control: pNIC-Bsa4 Uncut
Lane 3: Control: pNIC-Bsa4 Cut with HincII
Lane 4: alpha-1 pNIC-Bsa4 with Insert Cut with HincII
Lane 5: alpha-2 pNIC-Bsa4 with Insert Cut with HincII
Lane 6: alpha-3 pNIC-Bsa4 with Insert Cut with HincII
Lane 7: alpha-4 pNIC-Bsa4 with Insert Cut with HincII
Lane 8: beta-2 pNIC-Bsa4 with Insert Cut with HincII
Lane 9: beta-3 pNIC-Bsa4 with Insert Cut with HincII
Lane 10: tau-1 pNIC-Bsa4 with Insert Cut with HincII
The Restriction Enzyme Digest positively confirms that the full Insert has been annealed to the accepting vector. Unfortunately, the gel was not run long enough (20 minutes, 130 V), resulting in a lackluster image. Fortunately, the digests on the Control (Lane 3) and the samples are distinguishable. There should be 3 cuts above 500 bp in the Control vector (Lane 3) according to the virtual digest available from NEBcutter. The top most band on Lane 3 may be the result of DNA sample from Lane 2 or DNA that was not cut. Meanwhile, it is also theorized that the pNIC-Bsa4 with insert will have 5 cuts, source: virtual digest from NEBcutter. What was expected correlates to the actual result, especially in alpha-2 and alpha-4 (Lane 4 and Lane 6, respectively). Overall, difference in band sizes indicates a successful transformation of pNIC-Bsa4. The next step is to verify the quality of the DNA amplification via DNA Sequencing.
pNIC-Bsa4 No Insert Virtual Digest (NEBcutter 2)
pNIC-Bsa4 DOXP Virtual Digest (NEBcutter 2)
Nanodrop of Miniprepped pNIC-Bsa4 Insert
Originally, we grew the E. Coli in 8 separate transformation tubes (alpha-1, alpha-2, alpha-3, alpha-4, beta-1, beta-2, beta-3, tau-1). Unfortunately, beta-1 had better things to do and died away, rest in peace. The alpha and beta colonies grew on two plates that contained sample B (48 ul Accepting Vector, 16 ul Insert) while the tau colony grew on a plate that contained sample A (2 ul Accepting Vector, 4 ul Insert). Only one colony grew from sample A (tau-1) while 15-20 colonies grew from sample B. The nanodrop results above indicate a very high concentration of pNIC-Bsa4 with Insert, showing promise for yield considerations. Additionally, the 260/230 and 260/280 absorbance values are in the recommended range, though higher 260/230 values would be more optimistic.
Another note: During the transformation step, 15 minutes passed by before SOC media was added after 30 minutes in ice. Additionally, the time in the shaking incubator was cut to 40 minutes.
September 11, 2012
Accepting vector pNIC-Bsa4 digest verification with Gel Electrophoresis
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: pNIC-Bsa4 cut with BsaI (AB)
Lane 4: Skip
Lane 5-6: pNIC-Bsa4 cut with BsaI (KR)
Lane 7-10: Skip
PCR Clean Up pNIC-Bsa4
260/280 ratio is 1.85, 260/230 ratio is 2.29. Concentration is 49.8 ng/ul. Sample is usable as an accepting vector.
260/280 ratio is 1.90, 260/230 ratio is 2.34. Concentration is 68.1 ng/ul. Sample is usable as an accepting vector.
Week 1
September 07, 2012
PCR^2 cleanup Nanodrop
Absorbance ratios are 1.89 at 260/280 (indicative of pure DNA) and 1.99 at 260/230 (another indication of pure DNA). Concentration is 105.0 ng/ul, suitable for inserting vector preparation and bacteria transformation.
Gel check of all PCR samples to determine viability.
Lane 1: Primary alpha - PCR done on 09.06.12
Lane 2: Primary gamma - PCR done on 09.06.12
Lane 3: Primary alpha - PCR done on 09.04.12
Lane 4: Primary beta - PCR done on 09.04.12
Lane 5: Primary gamma - PCR done on 09.04.12
Lane 6: Primary Sample A
Lane 7: Primary Sample C
Lane 8: Secondary gamma - PCR done on 09.06.12
Lane 9: Secondary from 08/01 sample
Lane 10: Secondary from 07/18 sample
All samples seem to be okay, other than Lane 9 DNA sample.
September 06, 2012
Gel check of secondary PCR^2 from September 04, 2012 - unsuccessful. Samples were left at 4 degrees Celsius.
Tested Q5 High Fidelity Hot Start Polymerase with recommended NEB guidelines for primary PCR (20 cycles) - successful.
Secondary PCR with Q5 HF was done (KOD thermocycling protocol) - successful.
Lane 1: skip
Lane 2: 1 kb DNA Ladder
Lane 3-6: secondary PCR^2 (5 ul) KOD protocol
Lane 7-8: skip
Lane 9: ADO primary
Lane 10: ADO secondary
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: MM primary PCR
Lane 4: MM secondary PCR
Lane 5: KR primary PCR (alpha)
Lane 6: KR primary PCR (beta)
Lane 7: KR primary PCR (gamma)
Lane 8: skip
Lane 9: skip
Lane 10: skip
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: PCR^2 Sample ?
Lane 4: PCR^2 Sample ?
Lane 5-10: Skip
September 04, 2012
Tested Q5 High Fidelity Hot Start Polymerase with KOD thermocycling protocol. Primary (30 cycles) and Secondary PCR were done.
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: Primary PCR alpha (06/18/12) (5 ul)
Lane 4: Primary PCR beta (07/03/12) (5 ul)
Lane 5: Primary PCR gamma (08/01/12) (5 ul)
Lane 6-10: Skip
Lane 1: Skip
Lane 2: 1 kB DNA Ladder (5 ul)
Lane 3: Secondary PCR alpha (06/18/12) (5 ul)
Lane 4: Secondary PCR beta (07/03/12) (5 ul)
Lane 5: Secondary PCR gamma (08/01/12) (5 ul)
Lane 6-10: Skip
Both overlap extension PCR worked, indicating that the KOD protocol (slightly modified with higher denaturation temp. at 98 Centigrade, and higher extension temp. at 72 Centigrade. Lane 3 in the Secondary PCR trial failed, most likely to negligence when some sample was lost before thermocycling.
August 29, 2012
Tested Q5 High Fidelity Polymerase (No hot start) with thermocycle protocol given by the info. booklet.
August 02, 2012
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Alpha primary PCR
Lane 4: Alpha secondary PCR
Lane 5: Beta primary PCR
Lane 6: Beta secondary PCR
Lane 7: Gamma primary PCR
Lane 8: Gamma secondary PCR
Gel results above indicate poor amplification of DNA. Other researchers experienced poor results also, indicating the KOD polymerase was not of usable quality anymore.
August 01, 2012
All 12 samples did not contain DXR recombinant gene. Only tail-end primers were sequenced, indicating contamination by smaller DNA fragments.
July 30, 2012
Submitted 12 samples from July 27, 2012 to DNA Sequencing
July 27, 2012
Restriction Enzyme PvuII Digest of Miniprep Samples
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Control pNIC-Bsa4 No Insert
Lane 4: pNIC-Bsa4 A3 No.1 Insert
Lane 5: pNIC-Bsa4 A4 No.2 Insert
Lane 6: pNIC-Bsa4 A4 No.3 Insert
Lane 7: pNIC-Bsa4 B4 No.4 Insert
Lane 8: pNIC-Bsa4 A3 No.5 Insert
Lane 9: pNIC-Bsa4 B4 No.6 Insert
Lane 10: Skip
Star Activity is visible in Lanes 9&10.
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Control pNIC-Bsa4 No Insert
Lane 4: pNIC-Bsa4 A4 No.7 Insert
Lane 5: pNIC-Bsa4 A4 No.8 Insert
Lane 6: pNIC-Bsa4 A3 No.9 Insert
Lane 7: pNIC-Bsa4 B3 No.10 Insert
Lane 8: pNIC-Bsa4 B3 No.11 Insert
Lane 9: pNIC-Bsa4 A3 No.12 Insert
Lane 10: Skip
Star Activity is visible in Lanes 6 thru 9.
Incubated for 3 hours at 37 degrees Celsius. No heat-block treatment past the water-block. This may explain the star activity in several of the lanes above.
July 26, 2012
Centrifuged and retrieved 12 pellets. We purified the contents of the lysate and measured the concentrations, below.
A3 No. 1 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 86.7 ng/ul, 260/280 1.99 260/230 1.89
A4 No. 2 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 78.5 ng/ul 260/280 1.82 260/230 1.14
A4 No. 3 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 55.4 ng/ul 260/280 2.03 260/230 1.79
B4 No. 4 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 53.1 ng/ul 260/280 1.86 260/230 1.71
A3 No. 5 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 92.2 ng/ul 260/280 1.90 260/230 1.64
B3 No. 6 50 ul Tris-HCL - Nanodrop pNIC-Bsa4, Concentration 63.0 ng/ul 260/280 1.78 260/230 1.22
A4 No.7 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 122.3 ng/ul, 260/280 1.77 260/230 1.02
A4 No.8 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 76.2 ng/ul, 260/280 1.82, 260/230 1.16
A3 No. 9 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 66.4 ng/ul 260/280 2.00 260/230 1.58
B3 No. 10, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 79.0 ng/ul, 260/280 1.85, 260/230 1.24
B3 No. 11, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 80.6 ng/ul 260/280 1.92, 260/230 1.48
A3 No. 12, 50 ul Nanopure H20 - Nanodrop pNIC-Bsa4, Concentration 60.4 ng/ul 260/280 2.02, 260/230 1.91
July 25, 2012
Made master-plate and 12 tubes to incubate for 16 hours.
July 24, 2012
Prepared 15*1ml aliquots 1 mg/ml Kanamycin Stock. Few colonies are visible on the four plates grown yesterday (July 23, 2012)
July 23, 2012
Annealed and Transformed PCR product 3 (214) and PCR product 4 (170) with newly cut pNIC-Bsa4 Vector (?) and AH pNIC-Bsa4 Vector.
July 20, 2012
Restriction Enzyme Digest, Verification of PCR Product and Accepting Vector, More pNIC-Bsa4 cut
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: PCR^2 AH
Lane 4: 1A Miniprep Sample July 18, 2012
Lane 5: 1B Miniprep Sample July 18, 2012
The stream of DNA in Lane 4 and Lane 5 indicate that we have ended up with extraneous DNA.
To prepare for another trial run, we analyzed newly made inserting PCR product and accepting vector.
Lane 1: Skip
Lane 2: 1kb Ladder
Lane 3: PCR Clean Up 3?
Lane 4: PCR Clean up 4?
Lane 5: 072012 pNIC-Bsa4 Cut Vector
Lane 6: 1A Miniprep Sample July 18, 2012
Lane 7: 1B Miniprep Sample July 18, 2012
pNIC-Bsa4 cut (started at 133 ng/ul) concentration, 27.1 ng/ul
July 19, 2012
Annealing and Transformation Take 2, PCR strikes back
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: AB pNIC Cut
Lane 4: AB pNIC Cut
Lane 5: Skip
Lane 6: KR Secondary PCR
Nanodrop concentration: 213.9 ng/ul (eluted in 30 ul 10mM Tris HCl)
Nanodrop concentration: 170.2 ng/ul (eluted in 30 ul 10 mM Tris HCl)
We also submitted PCR Insert and pNIC-Bsa4 accepting vector (July 16, 2012 and July 17, 2012) after cohesive end generation to DNA sequencing to determine whether cohesive ends had been made before annealing and transformation.
July 18, 2012
PCR^2 Clean Up, Gel Analysis of Cut vector and PCR Insert (before and after cohesive ends).
Nanodrop P.fal after PCR Clean Up 1A - Concentration 76.8 ng/ul, Absorbance ratios indicate pure DNA. Contaminated
Nanodrop P.fal after PCR Clean Up 5B - Concentration 71.5 ng/ul, Absorbance ratios indicate pure DNA. Contaminated
July 27, 2012. The samples above did not contain the insert. The absorbance spectrums most likely indicate the presence of chromosomal DNA.
Lane 1: Skip
Lane 2: AB pNIC-Bsa4 Cut
Lane 3: AB pNIC-Bsa4 Cut
Lane 4: Skip
Lane 5: PCR Clean Up 3? KR
Lane 6: PCR Clean Up 4? KR
Lane 7: pNIC-Bsa4 Cut (after cohesive end generation)
Lane 8: PCR insert (after cohesive end generation)
Gel above revealed that PCR product and accepting vector were 'usable' - but does not demonstrate if cohesive ends had been formed.
July 17, 2012
Re Digest No. 2 with PvuII and Cohesive End Gel analysis
Lane 1: 1 kb DNA Ladder
Lane 2: AB pNIC Cut
Lane 3: pNIC-Bsa4 (Uncut, no Insert)
Lane 4: pNIC-Bsa4 (Cut with PvuII, no Insert)
Lane 5-9: pNIC-Bsa4 (Cut with PvuII, with insert)
We ran the restriction digest again to verify the findings from July 16, 2012. The results from DNA sequencing indicate no matches with the gene of interest.
July 16, 2012
RE Digest with PvuII and DNA Sequencing
Lane 1: skip
Lane 2: 1kb DNA Ladder
Lane 3: pNIC-Bsa4 (no PvuII cut, no insert)
Lane 4: pNIC-Bsa4 (after PvuII cut, no insert)
Lane 5-9: pNIC-Bsa4 (after PvuII cut, with insert)
Lane 10: skip
Unexpectedly, there are no bands in Lanes 5-9. This indicates that vector with the gene insert had not been transformed, even though we witnessed colony growth on the master plate.
Samples were submitted for DNA sequencing.
July 13, 2012
Miniprep to extract and purify plasmid DNA from tubes
2 ul Sample 2 at 260 nm. Contaminated
2 ul Sample 2 Trial 2 at 260 nm. Contaminated
2 ul Sample 4 at 260 nm. Contaminated
2 ul Sample 4 Trial 2 at 260 nm. Contaminated
2 ul Sample 6 at 260 nm. Contaminated
2 ul Sample 6 Trial 2 at 260 nm. Contaminated
2 ul Sample 7 at 260 nm. Contaminated
2 ul Sample 7 Trial 2 at 260 nm. Contaminated
2 ul Sample 8 at 260 nm. Contaminated
2 ul Sample 8 Trial 2 at 260 nm. Contaminated
Only colonies in tubes 2,4,6,7,8 produced a pellet after we centrifuged the conical tubes and removed supernatant. This is surprising since all tubes were incubated in similar conditions. We may have chosen 'bad' colonies (colonies were too large) in tubes 1,3,5. After plasmid preparation, the plasmid was eluted in 50ul Elution Buffer. The nanodrop results had concentrations between 25 ng plasmid/ul to 120 ng plasmid/ul. July 27, 2012. None of the samples contained the insert. The high 260/230 absorbance ratios may indicate that samples have been contaminated. The samples in this trial were chosen from plates that contained many ( ~100 to 200 colonies), indicating that the kan. did not kill cells that did not transform.
July 12, 2012
Protein Purification of FtHAP, Master Plate Growth of P.fal. Dxfr
Elution 1 FtHAP
Elution 2 FtHAP
The nano drop measurements reveal very low absorbance values of protein. This is not surprising, as we deviated from protocol often.
8 colonies from the 2 plates transformed with the pNIC-Bsa4 vector were selected to grow in 8 tubes and one agar plate at 37 degrees Celsius.
July 11, 2012
Gel Check of pNIC-Bsa4 cut with BsaI after PCR clean-up
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: 2.5 ul pNIC-Bsa4 digested with BsaI+ 0.5 ul blue dye
Lane 4-10: Skip
The two bands indicate two cuts were made, indicative of good result. Consequently, we made cohesive ends for the accepting vector (pNIC-Bsa4) and the inserting PCR DNA product (P.fal gene). The two pieces were annealed and transformed in 25 ul of DH5a Competent Cells and plated (2 plates) for overnight incubation.
July 10, 2012
Nanodrop pNIC-Bsa4
We cut the plasmid with the Bas1 Restriction Enzyme for 3 hours at 37 degrees Celsius. After PCR-Clean Up, we measured the concentration of plasmid - 40.8 ng/ul.
July 09, 2012
Nanodrop P.fal. DXFR (Sample A)
We did PCR Clean Up using the PCR^2 aliquots and measured the concentration of the DNA from scratch using the spectrophotometer. The average concentration (n=2) is 71.25 ng/ul.
July 06, 2012
Secondary PCR and PCR^2
Lane 1: Skip Lane 1: Skip
Lane 2: 1kb DNA Ladder Lane 2: 1kb DNA Ladder
Lane 3: PCR^2 Aliquot 1 A Lane 3: PCR^2 Aliquot 1 C
Lane 4: PCR^2 Aliquot 2 A Lane 4: PCR^2 Aliquot 2 C
Lane 5: PCR^2 Aliquot 3 A Lane 5: PCR^2 Aliquot 3 C
Lane 6: PCR^2 Aliquot 4 A Lane 6: PCR^2 Aliquot 4 C
Lane 7: Skip Lane 7-10: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Lane 1: Skip
Lane 2: 1kb DNA Ladder
Lane 3: Secondary PCR A
Lane 4: Secondary PCR C
Lane 5: Secondary PCR C (Sample tube was crushed in the thermocycler, also prematurely stopped final elongation).
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Both Secondary PCR and PCR^2 have worked.
July 05, 2012
Primary and Secondary PCR
Lane 1: Skip
Lane 2: 100 bp DNA Ladder
Lane 3: 1kb DNA Ladder
Lane 4: Primary PCR (PA)
Lane 5: Secondary PCR Option B (PA)
Lane 6: Skip
Lane 7: Primary PCR, old Oligo Mix A (KR)
Lane 8: Primary PCR, new Oligo Mix B (KR)
Lane 9: Primary PCR, new Oligo Mix C (KR)
Lane 10: Skip
(Continuing from July 02, 2012)
Comparing Lane 8 and Lane 9, Oligo Mix B was not properly prepared. Consequently, Oligo Mix B was discarded for future tests. The similarity in DNA product in Lane 7 and Lane 9 suggests that the old Oligo Mix was correctly prepared, but mistakes were made while preparing Secondary PCR.
July 03, 2012
Remade Oligo-Mix (two separate batches).
July 02, 2012
Lane 1: Skip
Lane 2: 1kb DNA Ladder, 5 ul
Lane 3: Secondary PCR Option B, 15 ul
Lane 4: Skip
Lane 5: Skip
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
(Continuing from June 29, 2012)
The amplified Gene of Interest DNA is faintly visible in Lane 3. From the line matching up with the 1.5k bp, the band dims. Even though the band should be intense, it may be enough to validate the visible band from June 29, 2012.
June 29, 2012
Looks good - hopefully the next round turns out better. - Dr. B
Lane 1: Skip
Lane 2: Skip
Lane 3: 1kb DNA Ladder
Lane 4: Primary PCR or Secondary PCR Option B? [KR]
Lane 5: Primary PCR or Secondary PCR Option B? [KR]
Lane 6: Skip
Lane 7: Skip
Lane 8: 100 bp DNA Ladder [UM]
Lane 9: primary PCR [UM]
Lane 10: secondary PCR B [UM]
(Continuing from June 27, 2012)
In this attempt to clone the gene with custom primers, we changed the annealing temperature to 59 degrees celsius, diluted the custom primers in 10 ul final volume (previously made 5 ul final volume), and stored the PCR product in the -20 fridge immediately after the PCR cycling finished. However, the samples did not fluoresce well, evident above. Assuming the band in lane 3 is the 3kb DNA ladder fragment, then the band in lane 4 may be 1500 bp, which is the size of the gene we are studying.
We also monitored growth and harvesting of competent BL21 cells for protein expression.
June 28, 2012
Made 10x 50 ml and 5x 500 ml LB media for growing bacteria.
June 27, 2012
Lane 1: 1 kb DNA Ladder
Lane 2: primary PCR [AH]
Lane 3: secondary PCR [AH]
Lane 4: secondary PCR B new (but old custom primers) [KR]
Lane 5: secondary PCR B new (but new custom primers) [KR]
Lane 6: secondary PCR A new (but old Oligo Mix primers) [KR]
Lane 7: secondary PCR A new (but new Oligo Mix primers) [KR}
Lane 8: secondary PCR A old [KR]
Lane 9: primary PCR new [KR]
Lane 10: secondary PCR B old [KR]
(Continuing from June 26, 2012)
None of the PCR products fluoresced under UV. The faint stream in Lane 9 indicates that Primary PCR (with a newer sample of custom oligonucleotides) may have worked, but it is pale in comparison to Alex H. Primary PCR streak in Lane 2, indicating that it is not accurate.
June 26, 2012
Secondary PCR Overlap Option B
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: Skip
Lane 4: Secondary PCR (Option B) KR
Lane 5: Skip
Lane 6: Skip
Lane 7: Skip
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
(Continuing from June 22, 2012)
Despite increasing the annealing temperature to 65 degrees Celsius, there is no resultant amplified DNA (would have been visible in Lane 4). This indicates that the product was not amplified. Either the template (primary PCR product) or the custom primers may be erroneous. (June 27, 2012) Annealing temperature is not 65 degrees Celsius; should be 60 degrees Celsius. See June 22, 2012.
June 25, 2012
PCR pNIC-Bsa4
Primers: pLIC-for and pLIC-rev
Lane 1: Skip
Lane 2: 100 bp DNA ladder 5 ul
Lane 3: ? ng pNIC-Bsa4 KRAB
Lane 4: ? ng pNIC-Bsa4 KRAB
Lane 5: Control KRAB
Lane 6: Skip
Lane 7: ? ng pNIC-Bsa4 AH
Lane 8: ? ng pNIC-Bsa4 AH
Lane 9: ? ng pNIC-Bsa4 AH
Lane 10: Control AH
Sample No. 3 KRAB (smallest amount of pNIC-Bsa4) is not shown above because it was misplaced after being taken out of the PCR machine.
June 22, 2012
Secondary PCR Overlap Option B
Lane 1: Skip
Lane 2: 1 kb DNA Ladder
Lane 3: 100 bp DNA Ladder
Lane 4: Primary PCR AB
Lane 5: Secondary PCR (Option A) AB
Lane 6: Secondary PCR (Option B) AB
Lane 7: Secondary PCR (Option B) KR
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
Sample (prepared on June 21, 2012) was stored overnight at 4 degrees celsius. After electrophoresis the gel didn't want to get analyzed, so it slipped onto the ground. We then used 15 ul of the same sample on another gel, above. However, the DNA in this Lane 7 forgot to fluoresce. The annealing temperature may be suspect. We did not change the binding temperature from the Primary PCR. Since the melting temperature of the pNIC-Bsa4 custom primers is approximately 70 degrees Celsius, an annealing temperature of 65 degrees Celsius may yield better results. (June 27, 2012) The previous statement is incorrect. We added 3 ul of 25 mM MgSO4 into 50 ul total volume, resulting in 1.5 mM MgSO4. The melting temperature of the custom primers is about 65 degrees Celsius at this concentration of Mg. The correct annealing temperature is around 60 degrees Celsius.
062112- Kaarthik. Nice job. Can you add a caption specifying the ladder size, your predicted gene size, and what the two different lanes are.
Thanks, Dr. B
June 20, 2012
Primary PCR Overlap and Secondary PCR Overlap Option A
Lane 1: Skip
Lane 2: Skip
Lane 3: 1 kb DNA Ladder
Lane 4: Primary PCR AB
Lane 5: Secondary PCR (Option A) AB
Lane 6: Primary PCR KR
Lane 7: Secondary PCR (Option A) KR (Approx. 1.5 kb).
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
The smear from the primary PCR is visible on Lane 6 and the secondary PCR product band is visible on Lane 7. It has a size of 1.5 kb. The actual gene size is 1467 bp.
June 20, 2012
PCR pNIC-Bsa4
Primers: pLIC-for and pLIC-rev
Lane 1: 100 bp DNA ladder 5 ul
Lane 2: 0.00824 ng pNIC-Bsa4 DB
Lane 3: 0.0824 ng pNIC-Bsa4 DB
Lane 4: 0.824 ng pNIC-Bsa4 DB
Lane 5: 0.00824 ng pNIC-Bsa4 KR
Lane 6: 0.0824 ng pNIC-Bsa4 KR
Lane 7: 0.824 ng pNIC-Bsa4 KR
Lane 8: Skip
Lane 9: Skip
Lane 10: Skip
We used an annealing temperature of 48 degrees Celsius.
June 19, 2012
Taking a break.
June 18, 2012
Made Oglio-Mix.
June 15, 2012
Nanodrop pGBR22
June 15, 2012
PCR pmCherry using VDS Primers and M13 Primers
Lane 1: Skip
Lane 2: 100 bp DNA ladder 5 ul
Lane 3: 0.018 ng pmCherry VDS Set
Lane 4: 0.18 ng pmCherry VDS Set
Lane 5: 1.8 ng pmCherry VDS Set
Lane 6: Skip
Lane 7: 0.018 ng pmCherry M13 Set
Lane 8: 0.18 ng pmCherry M13 Set
Lane 9: 1.8 ng pmCherry M13 Set
Lane 10: Skip
There should be a dark distinct band instead of a faint band since Lane 9 contained 1.8 ng pmCherry. Maybe it jumped over to Lane 10.
June 14, 2012
Primer Design
We designed a forward and reverse primer that will allow us to PCR the P.fal. gene and insert it into an expression vector (pNIC-Bsa4).
Forward Primer:
5’ TACTTCCAATCCATGAAAAAGTATATCTACATCTAC 3’ 36 bp
GC Content 30.6%
0 mM Mg2+ Tm 56.7 oC 1.5 mM Mg2+ Tm 64.9 oC 2 mM Mg2+ Tm 65.4 oC
4 mM Mg2+ Tm 66.5 oC 6 mM Mg2+ Tm 67.1 oC
Reverse Primer:
5’ AAACACAACTCTTCTTGA 3’ Reverse complement it:
5’ TATCCACCTTTACTGTCAAGAAGAGTTGTGTTT3’ 33 bp
GC Content 36.4%
0 mM Mg2+ Tm 59.7 oC 1.5 mM Mg2+ Tm 67.6 oC 2 mM Mg2+ Tm 68.1 oC
4 mM Mg2+ Tm 69.2 oC 6 mM Mg2+ Tm 69.7 oC
June 13, 2012
PCR of pGBR22 using M13 Primers
We repeated the experiment from June 07, 2012 but used the M13 Primer Set instead of the SP6/T7 Primer Set.
Lane 1: Skip
Lane 2: 100bp DNA ladder
Lane 3: 0.3 ng (1% conc.) pGBR22 plasmid KR
Lane 4: 3 ng (1% conc.) pGBR22 plasmid KR
Lane 5: 30 ng (10% conc.) pGBR22 plasmid KR
Lane 6: Control. No pGBR22 plasmid
Lane 7: 0.3 ng (1% conc.) pGBR22 plasmid AB
Lane 8: 3 ng (1% conc.) pGBR22 plasmid AB
Lane 9: 30 ng (10% conc.) pGBR22 plasmid AB
Lane 10: Control. No pGBR22 plasmid. Band maybe contamination from Lane 9.
June 11, 2012
Transformation of DH5alpha cells with pGFP
NOTE: PLATE A AND C WERE REVERSED
Plate A: 25ng of Plasmid, 224 colonies/ng
Plate B: 5ng of Plasmid, 800 colonies/ng
Plate C: 1ng of Plasmid, 1200 colonies/ng
June 07, 2012
Amplify Purple Protein coding sequence in the pGBR22 plasmid using Forward and Reverse Primers
PCR of pGBR22 using SP6/T7 Primers
Lane 1: Skip
Lane 2: 100bp DNA ladder
Lanes 3-8: PCR Samples of pGBR22 using SP6/T7 Primers. No fragment bands are visible.
We may have erred in pipetting or stoichiometric calculations. One plausible reason for the absence of any DNA fragments may be incorrect thermocycling programming (the annealing temperature entered corresponded to the M13 primer set, not the SP6/T7 primer set).
June 07, 2012
Restriction Digest Result Gel:
Lane 1: Skipped
Lane 2: DNA Ladder
Lane 3: Uncut Plasmid AB
Lane 4: EcoRI AB
Lane 5: PvuII AB
Lane 6: EcoR1+ PvuII AB
Lane 7: EcoRI KR
Lane 8: PvuII KR
Lane 9: EcoR1+ PvuII KR
Lane 10: Uncut Plasmid KR